Real Time Microarrays

ABSTRACT

This invention provides methods and systems for measuring the binding of analytes in solution to probes bound to surfaces in real-time.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application Nos.60/811,064, filed Jun. 5, 2006, and 60/840,060 filed on Aug. 24, 2006,which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Affinity-based biosensors exploit selective binding and interaction ofcertain bio-molecules (recognition probes) to detect specific targetanalytes in biological samples. The essential role of the biosensorplatforms and the parallel and miniaturized version of them asmicroarrays are to exploit specific bindings of the probe-targetcomplexes to produce detectable signals, which correlate with thepresence of the targets and conceivably their abundance. The essentialcomponents of such a system include the molecular recognition layer(capturing probes) integrated within or intimately associated with asignal-generating physiochemical transducer and a readout device.

To generate target-specific signal, the target analytes in the samplevolume generally first need to collide with the recognition layer,interact with the probes, bind to the correct probes, and ultimatelytake part in a transduction process. The analyte motion in typicalbiosensor settings (e.g., aqueous biological buffers) can be dominatedby diffusion spreading, which from a microscopic point of view is aprobabilistic mass-transfer process (modeled as a random walk for eachanalyte molecule). Accordingly, analyte collisions with probes become astochastic process. Moreover, because of the quantum-mechanical natureof chemical bond forming, the interaction between the probes and theanalytes molecules is also probabilistic, thus further contributing touncertainty and noise corruption of the measured data in biosensors andmicroarrays. We view such phenomena as inherent noise in the detectionsystem, which results in unavoidable uncertainties even when themeasurements are noiseless. Such inherent noise is essentiallyinevitable since it originates from the stochastic nature ofmolecular-level interactions. Its examples include Poisson noise sourcesin microarrays and image sensor detection shot-noise.

Beside the inherent noise, other non-idealities also corrupt the signalobtained by the microarray experiments. Examples of such phenomenainclude probe density variations, sample preparation systematic errors,and probe saturation. We define systematic errors as the unwanteddeviations from the intended detection procedure. If these errors areaccurately evaluated, in theory, they can be compensated by postexperiment data processing.

Gene expression microarrays are a widely used microarray platform. Thesesystems can measure the expression level of thousands of genessimultaneously, providing a massively-parallel affinity-based detectionplatform in life science research. Unfortunately, the uncertaintyoriginating from both inherent noise sources and systematic errors ineach experiment can obscure some of the important characteristics of thebiological processes of interest. The expression level uncertainty(overall measurement error) in microarrays, can originate from theprobabilistic characteristics of detection process as mentioned before,all the way from sample extraction and mRNA purification tohybridization and fluorescent intensity measurements. Currently, thereare various techniques which attempt to increase the accuracy andsignal-to-noise ratio (SNR) of the estimated values. Nonetheless, thesetechniques often rely on comparative methods, redundant spots, ormathematical algorithms which introduce confidence zones by excludingthe unreliable data and outliers. Independent of the method utilized,the degree in which the SNR can be improved in such approaches can stillbe limited by the inherent microarray noise and systematic errors.

The interfering signals originating from non-specific bindings inmicroarrays are generally referred to as “background signals.”Traditionally in microarray analysis, background signals and theirfluctuations are all considered as corruptive noise without any signalcontent. Users often implement a sub-optimal yet widely adoptedapproach. This technique defines a confidence threshold level for thesignal intensity in view of the background, which effectively dividesthe signals into irrelevant (below threshold) and relevant (abovethreshold) regimes. This particular approach is theoretically valid andoptimal only when there is a global background signal which is constanteverywhere. In practical microarray experiments, this assumption maynot, be not valid since the background and fluctuation level variesbetween spots. The approach can thus be sub-optimal. Even when localbackground subtraction methods are employed, the intensity data aresub-optimally processed, as the background signal that is present in theimmediate vicinity of a given microarray probe spot may not actually bethe same as the background signal from within the spot. The majoroutcome of background subtraction, regardless of the method that isused, is that the minimum detectable level (MDL) is higher thannecessary. It also contributes more errors in ratio analysis approaches,since low level signals are basically truncated away. Both of theseeffects in turn can reduce the microarray detection dynamic range.

Beside all the uncertainties within the measurement results, there isalso one major question in microarrays and all essentiallyaffinity-based biosensor systems, and that is of the necessaryincubation time (hybridization time for DNA microarrays). Since theincubation kinetics in the microarrays experiments is a function ofanalyte diffusion, reaction chamber size, temperature and bindingkinetics of every analyte species, as well as the unknown analyteconcentrations, the settling time of the system is quite complex andunpredictable. Although all these questions can, to some extent, beempirically addressed, they are still major impediments in microarraytechnology and platform-to-platform inconsistencies can be caused bythem.

In conventional fluorescent-based microarrays and other extrinsicreporter-based (label-based) biosensors assays, the detection ofcaptured analytes is usually carried out after the incubation step. Insome cases, proper fluorescent and reporter intensity measurements arecompromised in the presence of a large concentration of floating(unbound) labeled species in the incubation solution, whose signal canoverwhelm the target-specific signal from the captured targets. When theincubation is ceased and the solution is removed from the surface of thearray, the washing artifacts often occur that make the analysis of thedata even more challenging. Thus there exists a need for affinity basedsensors that are able to simultaneously obtain high quality measurementsof the binding characteristics of multiple analytes, and that are ableto determine the amounts of those analytes in solution.

SUMMARY OF THE INVENTION

One aspect of the invention is a method comprising measuring binding ofanalytes to probes on a microarray in real-time. In one embodiment, themethod comprises the steps of: contacting a fluid volume comprising aplurality of different analytes with a solid substrate comprising aplurality of different probes, wherein the probes are capable ofspecifically binding with the analytes; and measuring signals atmultiple time points while the fluid volume is in contact with thesubstrate, wherein the signals measured at multiple time points can becorrelated with the amount of binding of the analytes with the probes.One embodiment of the method further comprises using the signalsmeasured at multiple time points to determine the concentration of ananalyte in the fluid volume. In some embodiments, a change in thesignals with time correlates with the amount of the analytes bound tothe probes.

In some embodiments, the signals comprise electrochemical, electrical,mechanical, magnetic, acoustic, or electromagnetic signals. In someembodiments, the signals are electromagnetic signals comprisingfluorescence, absorption, or luminescence. In some embodiments, theanalytes comprise quenching moieties, and the electromagnetic signalsmeasured at multiple time points correlate, at least in part to thequenching of fluorescence. In some embodiments, the fluorescence that isquenched is due at least in part to fluorescent moieties bound to theprobes. In some embodiments, the fluorescence that is quenched is due atleast in part to fluorescent moieties bound to the solid substrate,wherein such fluorescent moieties are not covalently bound to theprobes. In some embodiments, the fluorescence that is quenched is due atleast in part to fluorescent moieties that are non-covalently bound tothe probe. In some embodiments, the optical signals measured at multipletime points are due, at least in part, to fluorescent resonance energytransfer (FRET).

In some embodiments, the different probes are located on the solidsubstrate at different addressable locations. In some embodiments thereare at least about 3, 4, 5 10, 50, 100, 500, 1000, 10,000, 100,000, or1,000,000 probes. In some embodiments, the solid substrate comprises oneor more beads.

In some embodiments, the analytes and/or the probes comprise thechemical species of nucleic acids or nucleic acid analogs, proteins,carbohydrates, or lipids. In some embodiments, the analytes and theprobes are the same type of chemical species. In some embodiments, theanalyte and the probe are each a different type of chemical species. Insome embodiments, the analyte and the probe comprise nucleic acids ornucleic acid analogs. In some embodiments, the probes comprise proteins,which in some cases can comprise antibodies or enzymes.

In some embodiments, the at least two time points are measured at a timewhen the amount of binding of an analyte to a probe corresponding tothat signal is less than 50% of saturation.

In some embodiments, two or more time points are measured when the fluidvolume is at one temperature, and two or more time points are measuredwhen the fluid volume is at a second temperature. In some embodiments,two or more time points are measured when the analyte in the fluidvolume is at one concentration, and two or more time points are measuredwhen the fluid volume is at a second concentration. In some embodiments,the concentration of the analyte is enriched between the sets of timepoints. In some embodiments, the concentration of the analyte is dilutedbetween the sets of time points.

In some embodiments, the solid substrate also comprises control spots.

In some embodiments, the method comprises the use of multiplefluorescent species, with different excitation and/or emission spectra.In some embodiments, the method comprises the use of multiple quencherspecies, with different quenching properties.

In some embodiments the method further comprises determining binding ofa probe with two or more analytes comprising subjecting binding datacorresponding to signal and time to an algorithm which determines thecontribution of each of the two or more analytes to the binding data. Insome embodiments, the probes comprise a fluorescent moiety and theinitial surface concentration of probes is determined by measuringfluorescence.

One aspect of the invention is a method of claim 2 wherein: (i) theanalytes comprise nucleic acid molecules labeled with a quencher; (ii)the probes comprise nucleic acid molecules; (iii) the substrate issubstantially planar and comprises an array of discrete locations atdifferent addresses on the substrate, wherein the locations haveattached thereto different probes and a fluorescent label that producesthe signal, wherein the fluorescent label is in sufficient proximity tothe probe whereby hybridization between an analyte and probe at thelocation causes FRET and/or quenching of the signal. In someembodiments, the fluorescent label is attached to the surface throughthe probe. In some embodiments, the fluorescent label is non-covalentlyattached to the probe. In some embodiments, the fluorescent label is notattached to the surface through the probe. In some embodiments, theconcentration of the analyte in the fluid based on the kinetics ofchange of the signal over time.

In some embodiments of the method of the invention, the fluid volumefurther comprises competitor molecules labeled with a quencher, whereinthe competitor molecules compete with the analytes for binding with theprobes.

One aspect of the invention is a method of detecting binding betweenanalyte molecules and an array of probe molecules comprising: (a)incubating the analyte molecules with the array of probe molecules,wherein binding between a analyte molecule and a probe molecule resultsin a change in a signal from the array; and (b) measuring the signalfrom the array over time during incubation. In some embodiments, bindingbetween a target molecule and a probe molecule results in a decrease ina signal from the array. In some embodiments, binding between a targetmolecule and a probe molecule results in an increase in a signal fromthe array.

One aspect of the invention is a method of detecting binding between ananalyte molecule and a probe comprising: (a) incubating an analytemolecule comprising a quencher of a donor-quencher pair with a probeimmobilized on a surface of a solid substrate under conditions wherebythe analyte molecule can bind to the probe, wherein the donor of thedonor-quencher pair is immobilized on the surface at a distance from theimmobilized probe and whereby binding between the analyte molecule andthe probe quenches a signal from the donor, and wherein the donor is notdirectly coupled to the immobilized probe; and (b) measuring the signal.In some embodiments, the method comprises incubating a plurality ofdifferent analyte molecules with a plurality of different probesimmobilized to different addressable locations on the surface of thesubstrate.

One aspect of the invention is a system comprising: (a) a device with(i) a solid support having a surface and (ii) a plurality of differentprobes, wherein the different probes are immobilized to the surface; (b)a fluid volume comprising an analyte wherein the fluid volume is incontact with the solid support, and (c) a detector assembly comprisingmeans to detect signals measured at multiple time points from each of aplurality of spots on the microarray while the fluid volume is incontact with the solid support. In some embodiments, the signalsmeasured at multiple time points detected by the detectors can becorrelated with the binding of analyte to the probes.

In some embodiments the system further comprises: (d) an assembly thatcontrols temperature of the solid support and/or the fluid volume. Insome embodiments, the different probes are immobilized at differentaddressable locations.

In some embodiments the system further comprises: (e) a data acquisitionsystem for acquiring and storing the data and (f) a computing system foranalyzing the signals. In some embodiments, the plurality of detectorscomprises an array of transducers. In some embodiments, the array oftransducers is capable of measuring an electrochemical, electrical,mechanical, magnetic, acoustic, or optical signal. In some embodiments,the solid substrate is in contact with the array of transducers. In someembodiments, the transducer array spaced away from the solid substrate.In some embodiments, one or more transducers correspond to anaddressable location. In some embodiments, the transducer array is anoptical transducer array which is optically coupled to the solidsubstrate. In some embodiments, the optical transducer array isoptically coupled to the solid substrate via one or more lenses.

In some embodiments, the system is capable of measuring a plurality ofbinding rates simultaneously. In some embodiments, the plurality ofbinding rates is used to determine the concentration of a plurality ofanalytes.

In some embodiments, the signal detected by the detector comprises anelectrochemical, electrical, mechanical, magnetic, acoustic, or opticalsignal. In some embodiments, the signal is generated from cyclicvoltammetry, impedance spectroscopy, or surface plasmon resonancesystems. In some embodiments, the signal is an optical signal. In someembodiments, the signal is from fluorescence, absorption, orluminescence. In some embodiments, the signals measured at multiple timepoints, correlating to the binding of analyte are due, at least in part,to fluorescent resonance energy transfer (FRET). In some embodiments,the signals measured at multiple time points, correlating to the bindingof analyte are due, at least in part, to quenching of a fluorescentsignal. In some embodiments, the signals measured at multiple timepoints are due, at least in part, to the interaction between an analytecomprising a quenching moiety, and probe comprising a fluorescentmoiety. In some embodiments, the moiety comprising the optical signal iscovalently bound to the probe molecule. In some embodiments, the moietycomprising the optical signal is non-covalently bound, to the probemolecule. In some embodiments, the moiety comprising the optical signalis bound to the probe molecule through another molecule. In someembodiments, the moiety comprising the optical signal is bound to thesubstrate.

One aspect of the invention is a system comprising: an assay assemblycomprising means to engage a microarray and means to perform an assay ona surface of the microarray; and a detector assembly comprising means todetect signals measured at multiple time points from each of a pluralityof spots on the microarray during the performance of the assay. In someembodiments, the means to perform the assay comprise means to provide acompartment wherein the surface of the microarray comprises a floor ofthe compartment and means to deliver reagents and analytes into thecompartment.

One aspect of the invention is a device comprising: a solid substratehaving a surface and a plurality of different probes, wherein thedifferent probes are immobilized to the surface at different addressablelocations, the addressable locations comprise optical signal moietiesbound to the surface, the optical signal moieties are not bound directlyto the probes, and the optical signal from the optical signal moietiesis capable of changing upon binding of an analyte to the probes. In someembodiments, the optical signal moiety comprises a dye, a luminescentmoiety, or a fluorescent moiety.

One aspect of the invention comprises software comprising: (a) acomputer executable code that accesses information about signalsmeasured at multiple time points at each of a plurality of knownlocations on a microarray, wherein the signal intensity is a function ofthe number of binding events between analyte molecules and probemolecules attached to the microarray at known locations. In someembodiments, the software further comprises: (b) code that executes analgorithm that uses the information to determine the expected number ofbinding events before binding has reached saturation, the existence andnumber of analyte molecules in the solution and the existence ofcross-hybridization. The algorithm furthermore can suppress the effectsof cross-hybridization on the acquired data.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows conventional detection procedure in affinity-basedbiosensors where capturing probes are used to capture target analyte inthe incubation phase, and detection is carried out in a dry-phase aftercompletion of the incubation

FIG. 2 shows conventional detection after completing incubation at timet₁ and the uncertainty associated with it.

FIG. 3 shows that in real-time microarray systems of the presentinvention, multiple measurement of the number of captured analytes canbe carried out, without the necessity of stopping the incubation step.

FIG. 4 shows a block diagram of the errors associated with conventionalDNA microarrays.

FIG. 5 shows a nucleic acid based real-time microarray system of thepresent invention where the probes are labeled with fluorescentmoieties.

FIG. 6 shows a nucleic acid based real-time microarray system of thepresent invention where the probes are labeled with fluorescentmoieties, the analytes are labeled with quenchers, and the fluorescentintensity on various spots can be used to measure the amount of analytespecifically bound to probe.

FIG. 7 shows a block diagram of a real-time microarray system of thepresent invention.

FIG. 8 shows an example of a real-time microarray system An example of areal-time microarray system where binding of BHQ2 quencher-labeled cDNAmolecules were detected using a fluorescent laser-scanning microscope.

FIG. 9 shows a real-time microarray system where the detection systemcomprises a sensor array in intimate proximity of the capturing spots.

FIG. 10 shows the layout of a 6×6 DNA microarray of the presentinvention

FIG. 11 shows a few samples of the real-time measurements of themicroarray experiment where control target analytes are added to thesystem

FIGS. 12-15 each show data for 4 different spots with similaroligonucletide capturing probes. The target DNA analyte is introduced inthe system at time zero and quenching (reduction of signal) occurs onlywhen binding happens.

FIG. 16 shows the signals measured during two real-time experimentswherein a target analyte (target 2) is applied to the microarray, at 2ng and at 0.2 ng.

FIG. 17 shows the signal versus time measured in a real-timeoligonucleotide array, and the fit of the data to an algorithm, where 80ng/50 μl of the target is applied to the array.

FIG. 18 shows he signal measured in a real-time oligonucleotide array,and the fit of the data to an algorithm, where 16 ng/50 μl of the targetis applied to the array.

FIG. 19 shows the results of a simulation indicating the potential ofsuppression of cross-hybridization over 3 orders of magnitude.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The methods, devices, and systems disclosed herein concern the real-timemeasurements of target binding events in microarrays and parallelaffinity-based biosensors. The methods and systems described herein canbe referred to as real-time microarray (RT-μArray) systems. Real-timemeasurement of the kinetics of multiple binding events allows for anaccurate and sensitive determination of binding characteristics or ofanalyte concentration for multiple species simultaneously. One aspect ofpresent invention is the evaluation of the abundance of target analytesin a sample by the real-time detection of target-probe binding events.In certain embodiments, RT-μArray detection systems measure theconcentration of the target analytes by analyzing the binding ratesand/or the equilibrium concentration of the captured analytes in asingle and/or plurality of spots. Some applications of RT-μArrays fallwithin the field of Genomics and Proteomics, in particular DNA andProtein microarrays. Some of the advantages of RT-μArray systems overconventional microarray platforms are in their higher detection dynamicrange, lower minimum detection level (MDL), robustness, and lowersensitivity to array fabrication systematic errors, analyte bindingfluctuation, and biochemical noise, as well as in the avoidance of thewashing step required for conventional microarrays.

One aspect of the invention is a method of measuring binding of analytesto a plurality of probes on surface in “real time”. As used herein inreference to monitoring, measurements, or observations of binding ofprobes and analytes of this invention, the term “real-time” refers tomeasuring the status of a binding reaction while that reaction isoccurring, either in the transient phase or in biochemical equilibrium.Real time measurements are performed contemporaneously with themonitored, measured, or observed binding events, as opposed tomeasurements taken after a reaction is fixed. Thus, a “real time” assayor measurement contains not only the measured and quantitated result,such as fluorescence, but expresses this at various time points, thatis, in hours, minutes, seconds, milliseconds, nanoseconds, etc. “Realtime” includes detection of the kinetic production of signal, comprisingtaking a plurality of readings in order to characterize the signal overa period of time. For example, a real time measurement can comprise thedetermination of the rate of increase or decrease in the amount ofanalyte bound to probe.

The measurements are performed in real time with a plurality of probesand analytes. The invention is useful for measuring probes and analytesthat bind specifically to one another. A probe and an analyte pair thatspecifically bind to one another can be a specific binding pair.

The methods and systems of the invention can be used for measuring thebinding of multiple specific binding pairs in the same solution at thesame time. In one embodiment of the invention a plurality of probeswhich are members of a specific binding pair are attached to a surface,and this surface is used to measure the binding kinetics of a pluralityof analytes which comprise the other member of the specific bindingpair.

One aspect of the invention is a method of measuring binding betweenanalyte and probe which lowers, and in some cases eliminates the noisewhich is present in conventional microarrays and which decreases thequality of the analyte-probe binding information. In conventionalmicroarrays and most of the affinity-based biosensors, the detection andincubation phases of the assay procedure are carried out at differenttimes. As shown in FIG. 1, conventional detection is carried out in adry-phase at the point where a scanning and/or imaging technique used toassess the captured targets.

The following analysis illustrates inherent problems with conventionalmicroarray analysis techniques, and the advantages of the real timemicroarray systems of the present invention in improving the quality ofthe binding measurement. Let x(t) denote the total number of capturedanalyte in a given spot of the microarray and/or affinity-basedbiosensor at a given time instant t. Furthermore, let x(t) denote theexpected value of x(t) when the incubation process has reachedbiochemical equilibrium. A typical microarray procedure is focused onestimating x(t), and using its value as an indication of the analyteconcentration in the sample; in fact, most data analysis techniquesdeduce their results based on x(t). Nevertheless, if we measure thenumber of captured analytes at time t₁ in the equilibrium, for any givenmicroarray spot it holds that x(t₁)≠x(t). This is due to the inherentbiochemical noise and other uncertainties of the system. This phenomenonis illustrated in FIG. 2, where the number of captured analytes in eachspot of the microarray fluctuates in time, even in biochemicalequilibrium. Accordingly, a single measurement taken at time t₁, whichis what conventional microarray experiments provide, essentially samplesa single point of the random process of analyte binding.

Now, consider the case where we are able measure x(t) multiple times, inreal-time without the necessity of stopping the incubation and analytebinding reaction. This platform, which we call the real-time microarrays(RT-μArrays), has many advantages over the conventional method. In someembodiments of RT-μArrays, the kinetic of the bindings can be observed.Therefore, one can test whether the system has reached biochemicalequilibrium or not. In other embodiments, multiple samples of x(t) aremeasured (see FIG. 3), and different averaging techniques and/orestimation algorithms can be used to estimate x(t) and othercharacteristics of process x(t).

FIG. 4 shows a block diagram of the errors associated with conventionalDNA microarrays. These may be categorized into three stages:pre-hybridization (steps 1 and 2), hybridization (step 3), andpost-hybridization (steps 4 and 5). The pre-hybridization errors arisefrom sample purification variations and the errors or variations inreverse transcribing mRNA to cDNA, in generating in vitro transcribedRNA complementary to the cDNA (cRNA, or IVT RNA), and or in labeling theanalytes (step 1), and the errors arising from non-uniform probespotting and or synthesis on the array (step 2). The hybridizationerrors arise from the inherent biochemical noise, cross-hybridization tosimilar targets, and the probe saturation (step 3). Post-hybridizationerrors include washing artifacts, image acquisition errors (step 4), andsuboptimal detection (step 5). The most critical of these are probedensity variations (step 2), probe saturation and cross-hybridization(step 3) and washing artifacts (step 4).

The methods and systems of the present invention can compensate for allthe above errors except for those of sample preparation (step 1). Probedensity variations can be measured prior to incubation and thereforeaccounted for in post-processing (step 5), incubation noise can bereduced by taking many samples (rather than a single one), as mentionedearlier, probe saturation can be avoided by estimating targetconcentrations from the reaction rates, and finally washing is avoidedaltogether.

One aspect of the invention is a method of measuring the binding of aplurality of analytes to a plurality of probes with a higher dynamicrange than obtained with conventional microarrays. In some embodimentsof the invention, the dynamic range is 3, 4, 5 or more orders ofmagnitude.

Methods

One aspect of the invention is a method comprising measuring binding ofanalytes to probes on a microarray in real-time. In one embodiment, themethod comprises the steps of: contacting a fluid volume comprising aplurality of different analytes with a solid substrate comprising aplurality of different probes, wherein the probes are capable ofspecifically binding with the analytes; and measuring signals atmultiple time points while the fluid volume is in contact with thesubstrate, wherein the signals measured at multiple time points can becorrelated with the amount of binding of the analytes with the probes.One embodiment of the method further comprises using the signalsmeasured at multiple time points to determine the concentration of ananalyte in the fluid volume.

In one embodiment the method involves the use of probe arrays in whicheach addressable location emits a signal that is quenchable upon bindingof an analyte. For example, the quenchable moiety (e.g., a fluorescentmoiety) is attached to the probe on the array or in close physicalproximity thereto. The surface of such array will emit signal from eachaddressable location which can be detected using, for example, amicroscope and a light detector (e.g., a CCD camera or CMOS imagesensor). The analytes in the sample are tagged with a quencher moietythat can quench the signal from the quenchable moiety. When the quencherdoes not, itself, emit a light signal, there is no signal from the fluidto interfere with the signal from the array. This diminishes the noiseat the array surface. During the course of a binding reaction betweenanalytes and substrate-bound probes, the signal at each addressablelocation is quenched. The signal at each addressable location ismeasured in real time, for example, by a CCD camera focused on the arraysurface. As the signal at any location changes as a result of bindingand quenching, the change is measured. These measurements over timeallow determination of the kinetics of the reaction which, in turn,allows determination of the concentration of analytes in the sample.

Alternatively if the analytes are labeled with a light-emittingreporter, such as a fluorescent label, signal at the surface of arrayresulting from binding of the labeled analyte molecules can be detectedby properly focusing the detector at the array surface, therebyminimizing the noise from signal in solution.

In another embodiment, the probes are attached to the surface of anarray comprising sensors, such as a CMOS sensor array, which produceelectrical signals that change as a result of binding events on theprobes. This also affords real time measurement of a plurality ofsignals on an array (Hassibi and Lee, IEEE Sensors journal, 6-6, pp.1380-1388, 2006, and Hassibi, A. “Integrated Microarrays,” Ph.D. ThesisStanford University, 2005.

Accordingly, the methods of this invention allow real time measurementsof a plurality of binding events on an array of probes on a solidsupport.

Analyte and Probe

The terms “probe” and “analyte” as used herein refer to molecularspecies that bind to one another in solution. A single probe or a singleanalyte is generally one chemical species. That is, a single analyte orprobe may comprise many individual molecules. In some cases, a probe oranalyte may be a set of molecules that are substantially identical. Insome cases a probe or analyte can be a group of molecules all of whichhave a substantially identical binding region. A “probe” and/or“analyte” can be any pair of molecules that bind to one another,including for example a receptor/ligand pair, or a hybridizing pair ofnucleic acids. In probes of the present invention are bound to a solidsurface. The analyte is in solution, and can also be referred to as thetarget or the target analyte. Thus, while the probe and analyte caninterchangeably be the different members of any binding pair, in somecases it is more advantageous for one or the other to be the probe orthe analyte, for instance where the molecule is more easily coupled tothe surface, it can be advantageous for that molecule to be the probe,or where a molecule is more soluble in the solution of interest, it canbe advantageous for that molecule to be the analyte.

The probes or analytes can be any type of chemical species. The probesor analytes are generally biomolecules such as nucleic acids, proteins,carbohydrates, lipids, or small molecules. The probe and analyte whichbind to one another can each be the same or different types of species.The analyte or probe may be bound to another type of molecule and maycomprise different molecules. For example, an analyte could be a proteincarbohydrate complex, or a nucleic acid connected to protein. Aprobe-analyte pair can also be a receptor-ligand pair. Where thechemical species is large or made of multiple molecular components, theprobe or analyte may be the portion of the molecule that is capable ofbinding, or may be the molecule as a whole. Examples of analytes thatcan be investigated by this invention include, but are not restrictedto, agonists and antagonists for cell membrane receptors, toxins andvenoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),hormone receptors, peptides, enzymes, enzyme substrates, substrateanalogs, transition state analogs, cofactors, drugs, proteins,antibodies, and hybridizing nucleic acids.

The term “probe” is used herein to refer to the member of the bindingspecies that is attached to the substrate. For instance, the probeconsists of biological materials deposited so as to create spottedarrays; and materials synthesized, deposited, or positioned to formarrays according to other current or future technologies. Thus,microarrays formed in accordance with any of these technologies may bereferred to generally and collectively hereafter for convenience as“probe arrays.” Moreover, the term “probe” is not limited to probesimmobilized in array format. Rather, the functions and methods describedherein may also be employed with respect to other parallel assaydevices. For example, these functions and methods may be applied withrespect to probe-set identifiers that identify probes immobilized on orin beads, optical fibers, or other substrates or media. The constructionof various probe arrays of the invention are described in more detailbelow.

In some embodiments, the probe and/or the analyte comprises apolynucleotide. The terms “polynucleotide,” “oligonucleotide,” “nucleicacid” and “nucleic acid molecule” as used herein include a polymericform of nucleotides of any length, either ribonucleotides (RNA) ordeoxyribonucleotides (DNA). This term refers only to the primarystructure of the molecule. Thus, the term includes triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. It also includes modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” include polydeoxyribonucleotides (containing2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), any othertype of polynucleotide which is an N- or C-glycoside of a purine orpyrimidine base, and other polymers containing nonnucleotidic backbones.A nucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925(1993) and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J.Am. Chem. Soc. 11 1:2321 (1989), O-methylphophoroamidite linkages (seeEckstein, Oligonucleotides and Analogues: A Practical Approach, OxfordUniversity Press), and peptide nucleic acid backbones and linkages (seeCarlsson et al., Nature 380:207 (1996)). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al., Proc. Natl. Acad.Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew.Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem.Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597(1994); Chapters 2 and 3, ASC Symposium Series 580, “CarbohydrateModifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook;Mesmaeker et al., Bioorganic & Medicinal Chem. Left. 4:395 (1994); Jeffset al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Left. 37:743(1996)) and non-ribose backbones, including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of labels, or to increase the stability and half-life ofsuch molecules in physiological environments.

In some embodiments of the invention, oligonucleotides are used. An“oligonucleotide” as used herein is a single-stranded nucleic acidranging in length from 2 to about 1000 nucleotides, more typically from2 to about 500 nucleotides in length. In some embodiments, it is about10 to about 100 nucleotides, and in some embodiments, about 20 to about50 nucleotides.

In some embodiments of the invention, for example, expression analysis,the invention is directed toward measuring the nucleic acidconcentration in a sample. In some cases the nucleic acid concentration,or differences in nucleic acid concentration between different samples,reflects transcription levels or differences in transcription levels ofa gene or genes. In these cases it can be desirable to provide a nucleicacid sample comprising mRNA transcript(s) of the gene or genes, ornucleic acids derived from the mRNA transcript(s). As used herein, anucleic acid derived from an mRNA transcript refers to a nucleic acidfor whose synthesis the mRNA transcript or a subsequence thereof hasultimately served as a template. Thus, a cDNA reverse transcribed froman mRNA, an RNA transcribed from that cDNA, a DNA amplified from thecDNA, an RNA transcribed from the amplified DNA, etc., are all derivedfrom the mRNA transcript and detection of such derived products isindicative of the presence and/or abundance of the original transcriptin a sample. Thus, suitable samples include, but are not limited to,mRNA transcripts of the gene or genes, cDNA reverse transcribed from themRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNAtranscribed from amplified DNA, and the like.

In some embodiments, where it is desired to quantify the transcriptionlevel (and thereby expression) of one or more genes in a sample, thenucleic acid sample is one in which the concentration of the mRNAtranscript(s) of the gene or genes, or the concentration of the nucleicacids derived from the mRNA transcript(s), is proportional to thetranscription level (and therefore expression level) of that gene.Similarly, it is preferred that the hybridization signal intensity beproportional to the amount of hybridized nucleic acid. While it ispreferred that the proportionality be relatively strict (e.g., adoubling in transcription rate results in a doubling in mRNA transcriptin the sample nucleic acid pool and a doubling in hybridization signal),one of skill will appreciate that the proportionality can be morerelaxed and even non-linear. Thus, for example, an assay where a 5 folddifference in concentration of the target mRNA results in a 3 to 6 folddifference in hybridization intensity is sufficient for most purposes.Where more precise quantification is required appropriate controls canbe run to correct for variations introduced in sample preparation andhybridization as described herein. In addition, serial dilutions of“standard” target mRNAs can be used to prepare calibration curvesaccording to methods well known to those of skill in the art. Of course,where simple detection of the presence or absence of a transcript orlarge differences of changes in nucleic acid concentration are desired,no elaborate control or calibration is required.

In the simplest embodiment, such a nucleic acid sample is the total mRNAor a total cDNA isolated and/or otherwise derived from a biologicalsample. The term “biological sample”, as used herein, refers to a sampleobtained from an organism or from components (e.g., cells) of anorganism. The sample may be of any biological tissue or fluid.Frequently the sample will be a “clinical sample” which is a samplederived from a patient. Such samples include, but are not limited to,sputum, blood, blood cells (e.g., white cells), tissue or fine needlebiopsy samples, urine, peritoneal fluid, and pleural fluid, or cellstherefrom. Biological samples may also include sections of tissues suchas frozen sections taken for histological purposes.

The nucleic acid (either genomic DNA or mRNA) may be isolated from thesample according to any of a number of methods well known to those ofskill in the art. One of skill will appreciate that where alterations inthe copy number of a gene are to be detected genomic DNA is preferablyisolated. Conversely, where expression levels of a gene or genes are tobe detected, preferably RNA (mRNA) is isolated.

Methods of isolating total mRNA are well known to those of skill in theart. For example, methods of isolation and purification of nucleic acidsare described in detail in Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993) and Chapter 3 of Laboratory Techniques inBiochemistry and Molecular Biology: Hybridization With Nucleic AcidProbes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed.Elsevier, N.Y. (1993)).

Frequently, it is desirable to amplify the nucleic acid sample prior tohybridization. One of skill in the art will appreciate that whateveramplification method is used, if a quantitative result is desired, caremust be taken to use a method that maintains or controls for therelative frequencies of the amplified nucleic acids.

In some embodiments, the probe and or the analyte may comprise apolypeptide. As used herein, the term “polypeptide” refers to a polymerof amino acids and does not refer to a specific length of the product;thus, peptides, oligopeptides, and proteins are included within thedefinition of polypeptide. This term also does not refer to or excludepost expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like. The“peptide” refers to polypeptides of no more than about 50 amino acids,while term “protein” refers to longer polypeptides, typically withthree-dimensional structures. Non-natural polypeptides containing one ormore analogs of an amino acid (including, for example, unnatural aminoacids, etc.), can also be useful in the invention, as can polypeptideswith substituted linkages, as well as other modifications known in theart, both naturally occurring and non-naturally occurring. Polypeptidesand proteins can have specific binding properties. For instance, anenzyme can have a region that binds specifically with a substrate,and/or has regions that bind to other proteins, such as the binding ofenzyme subunits. Antibodies, which can have very specific bindingproperties are also polypeptides.

In some embodiments the probe and/or analyte can comprise a carbohydratesuch as a polysaccharide. The term polysaccharide, as used herein,refers to a carbohydrate which is a polyhydroxy aldehyde or ketone, orderivative thereof, having the empirical formula (CH₂O)_(n) wherein n isa whole integer, typically greater than 3. Monosaccharides, or simplesugars, consist of a single polyhydroxy aldehyde or ketone unit.Monosaccharides include, but are not limited to, ribose, 2-deoxy-ribose,glucose, mannose, xylose, galactose, fucose, fructose, etc.Disaccharides contain two monosaccharide units joined by a glycosidiclinkage. Disaccharides include, for example, sucrose, lactose, maltose,cellobiose, and the like. oligosaccharides typically contain from 2 to10 monosaccharide units joined in glycosidic linkage. Polysaccharides(glycans) typically contain more than 10 such units and include, but arenot limited to, molecules such as heparin, heparan sulfate, chondroitinsulfate, dermatan sulfate and polysaccharide derivatives thereof. Theterm “sugar” generally refers to mono-, di- or oligosaccharides. Asaccharide may be substituted, for example, glucosamine, galactosamine,acetylglucose, acetylgalactose, N-acetylglucosamine,N-acetyl-galactosamine, galactosyl-N-acetylglucosamine,N-acetylneuraminic acid (sialic acid), etc. A saccharide may also resideas a component part of a larger molecule, for example, as the saccharidemoiety of a nucleoside, a nucleotide, a polynucleotide, a DNA, an RNA,etc.

In some embodiments, the analyte and/or probe is a small molecule.Generally the small molecule will be an organic molecule, for example,biotin or digoxigenin, but in some cases, the analyte can be inorganic,for example an inorganic ion such as lithium, sodium, ferric, ferrous,etc. The small molecule can also be an organometallic compound, havingboth inorganic and organic components.

Probes on a Solid Substrate

For the methods of the present invention, the probes are attached to asolid substrate. The solid substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides,semiconductor integrated chips etc. The solid substrate is preferablyflat but may take on alternative surface configurations. For example,the solid substrate may contain raised or depressed regions on whichsynthesis or deposition takes place. In some embodiments, the solidsubstrate will be chosen to provide appropriate light-absorbingcharacteristics. For example, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂SiN₄, modified silicon, or any one of a variety of gels or polymers suchas (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof. The solid support and thechemistry used to attach the solid support are described in detailbelow.

The substrate can be a homogeneous solid and/or unmoving mass muchlarger than the capturing probe where the capturing probes are confinedand/or immobilized within a certain distance of it. The mass of thesubstrate is generally at least 100 times larger than capturing probesmass. In certain embodiments, the surface of the substrate is planarwith roughness of 0.1 nm to 100 nm, but typically between 1 nm to 10 nm.In other embodiments the substrate can be a porous surface withroughness of larger than 100 nm. In other embodiments, the surface ofthe substrate can be non-planar. Examples of non-planar substrates arespherical magnetic beads, spherical glass beads, and solid metal and/orsemiconductor and/or dielectric particles.

For the methods of the present invention, the plurality of probes may belocated in one addressable region and/or in multiple addressable regionson the solid substrate. In some embodiments the solid substrate hasabout 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000,1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000,100,000-500,000, 500,000-1,000,000 or over 1,000,000 addressable regionswith probes.

In some embodiments it is also useful to have addressable regions whichdo not contain probe, for example, to act as control spots in order toincrease the quality of the measurement, for example, by using bindingto the spot to estimate and correct for non-specific binding.

Analyte/Probe Binding

The methods of the present invention are directed toward measuring thebinding characteristics of multiple probes and analytes in real time.The method is particularly useful for characterizing the binding ofprobes and analytes which specifically bind to one another. As usedherein, a probe “specifically binds” to a specific analyte if it bindsto that analyte with greater affinity than it binds to other substancesin the sample.

The binding between the probe and the analyte in the present inventionoccurs in solution. Usually the probe and analyte are biomolecules andthe solution is an aqueous solution. An aqueous solution is a solutioncomprising solvent and solute where the solvent is comprised mostly ofwater. The methods of the invention, however, can be used in any type ofsolution where the binding between a probe and an analyte can occur andbe observed.

Molecular recognition assays generally involve detecting binding eventsbetween two types of molecules. The strength of binding can be referredto as “affinity”. Affinities between biological molecules are influencedby non-covalent intermolecular interactions including, for example,hydrogen bonding, hydrophobic interactions, electrostatic interactionsand Van der Waals forces. In multiplexed binding experiments, such asthose contemplated here, a plurality of analytes and probes areinvolved. For example, the experiment may involve testing the bindingbetween a plurality of different nucleic acid molecules or betweendifferent proteins. In such experiments analytes preferentially willbind to probes for which they have the greater affinity. Thus,determining that a particular probe is involved in a binding eventindicates the presence of an analyte in the sample that has sufficientaffinity for the probe to meet the threshold level of detection of thedetection system being used. One may be able to determine the identityof the binding partner based on the specificity and strength of bindingbetween the probe and analyte.

The binding may be a receptor-ligand, enzyme-substrate,antibody-antigen, or a hybridization interaction. The probe/analytebinding pair or analyte/probe binding pair can be nucleic acid tonucleic acid, e.g. DNA/DNA, DNA/RNA, RNA/DNA, RNA/RNA, RNA. Theprobe/analyte binding pair or analyte/probe binding pair can be anucleic acid and a polypeptide, e.g. DNA/polypeptide andRNA/polypeptide, such as a sequence specific DNA binding protein. Theprobe/analyte binding pair or analyte/probe binding pair can be anynucleic acid and a small molecule, e.g. RNA/small molecule, DNA/smallmolecule. The probe/analyte binding pair or analyte/probe binding paircan be any nucleic acid and synthetic DNA/RNA binding ligands (such aspolyamides) capable of sequence-specific DNA or RNA recognition. Theprobe/analyte binding pair or analyte/probe binding pair can be aprotein and a small molecule or a small molecule and a protein, e.g. anenzyme or an antibody and a small molecule.

The probe/analyte binding pair or analyte/probe binding pair can be acarbohydrate and protein or a protein and a carbohydrate, a carbohydrateand a carbohydrate, a carbohydrate and a lipid, or lipid and acarbohydrate, a lipid and a protein, or a protein and a lipid, a lipidand a lipid.

The analyte/probe binding pair can comprise natural binding compoundssuch as natural enzymes and antibodies, and synthetic binding compounds.The probe/analyte binding pair or analyte/probe binding pair can besynthetic protein binding ligands and proteins or proteins and syntheticbinding ligands, synthetic carbohydrate binding ligands andcarbohydrates or carbohydrates and synthetic carbohydrate bindingligands, synthetic lipid binding ligands or lipids and lipids andsynthetic lipid binding ligands.

Nucleic Acid Systems

One particularly useful aspect of the present invention involvesspecific hybridization between an analyte and a probe, where bothcomprise nucleic acids.

As used herein an “oligonucleotide probe” is an oligonucleotide capableof binding to a target nucleic acid of complementary sequence throughone or more types of chemical bonds, usually through complementary basepairing, usually through hydrogen bond formation. The oligonucleotideprobe may include natural (i.e. A, G, C, or T) or modified bases(7-deazaguanosine, inosine, etc.). In addition, the bases inoligonucleotide probe may be joined by a linkage other than aphosphodiester bond, so long as it does not interfere withhybridization. Thus, oligonucleotide probes may be peptide nucleic acidsin which the constituent bases are joined by peptide bonds rather thanphosphodiester linkages. The oligonucleotide probes can also compriselocked nucleic acids (LNA), LNA, often referred to as inaccessible RNA,is a modified RNA nucleotide. The ribose moiety of the LNA nucleotide ismodified with an extra bridge connecting 2′ and 4′ carbons. The bridge“locks” the ribose in 3′-endo structural conformation, which is oftenfound in A-form of DNA or RNA. LNA nucleotides can be mixed with DNA orRNA bases in the oligonucleotide. Such oligomers are commerciallyavailable. The locked ribose conformation can enhance base stacking andbackbone pre-organization, and can increase the thermal stability(melting temperature) of oligonucleotides.

The term “nucleic acid analyte” or “target nucleic acid” or “target”refers to a nucleic acid (often derived from a biological sample andhence referred to also as a sample nucleic acid), to which theoligonucleotide probe specifically hybridizes. It is recognized that thetarget nucleic acids can be derived from essentially any source ofnucleic acids (e.g., including, but not limited to chemical syntheses,amplification reactions, forensic samples, etc.). It is either thepresence or absence of one or more target nucleic acids that is to bedetected, or the amount of one or more target nucleic acids that is tobe quantified. The target nucleic acid(s) that are detectedpreferentially have nucleotide sequences that are complementary to thenucleic acid sequences of the corresponding probe(s) to which theyspecifically bind (hybridize). The term target nucleic acid may refer tothe specific subsequence of a larger nucleic acid to which the probespecifically hybridizes, or to the overall sequence (e.g., gene or mRNA)whose abundance (concentration) and/or expression level it is desired todetect. The difference in usage will be apparent from context.

In the present invention, the specific hybridization of anoligonucleotide probe to the target nucleic acid can be measured inreal-time. An oligonucleotide probe will generally hybridize, bind, orduplex, with a particular nucleotide sequence under stringent conditionseven when that sequence is present in a complex mixture. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences.

For nucleic acid systems, the oligonucleotide probes of the presentinvention are designed to be complementary to a nucleic acid targetsequence, such that hybridization of the target sequence and the probesof the present invention occurs. This complementarity need not beperfect; there may be any number of base pair mismatches which willinterfere with hybridization between the target sequence and the singlestranded nucleic acids of the present invention. However, if the numberof mutations is so great that no hybridization can occur under even theleast stringent of hybridization conditions, the sequence is not acomplementary target sequence. Thus, an oligonucleotide probe that isnot substantially complementary to a nucleic acid analyte will nothybridize to it under normal reaction conditions.

The methods of the present invention thus can be used, for example, todetermine the sequence identity of a nucleic acid analyte in solution bymeasuring the binding of the analyte with known probes. The sequenceidentity can be determined by comparing two optimally aligned sequencesor subsequences over a comparison window or span, wherein the portion ofthe polynucleotide sequence in the comparison window may optionallycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical subunit (e.g.nucleic acid base or amino acid residue) occurs in both sequences toyield the number of matched positions, dividing the number of matchedpositions by the total number of positions in the window of comparisonand multiplying the result by 100 to yield the percentage of sequenceidentity.

The methods of the current invention when applied to nucleic acids, canbe used for a variety of applications including, but not limited to, (1)mRNA or gene expression profiling, involving the monitoring ofexpression levels for example, for thousands of genes simultaneously.These results are relevant to many areas of biology and medicine, suchas studying treatments, diseases, and developmental stages. For example,microarrays can be used to identify disease genes by comparing geneexpression in diseased and normal cells; (2) comparative genomichybridization (Array CGH), involving the assessment of large genomicrearrangements; (3) SNP detection arrays for identifying for SingleNucleotide Polymorphisms (SNP's) in the genome of populations; andchromatin immunoprecipitation (chIP) studies, which involve determiningprotein binding site occupancy throughout the genome, employingChIP-on-chip technology.

The present invention can be very sensitive to differences in bindingbetween nucleic acid species, in some cases, allowing for thediscrimination down to a single base pair mismatch. And because thepresent invention allows the simultaneous measurement of multiplebinding events, it is possible to analyze several speciessimultaneously, where each is intentionally mismatched to differentdegrees. In order to do this, a “mismatch control” or “mismatch probe”which are probes whose sequence is deliberately selected not to beperfectly complementary to a particular target sequence can be used, forexample in expression arrays. For each mismatch (MM) control in an arraythere, for example, exists a corresponding perfect match (PM) probe thatis perfectly complementary to the same particular target sequence. In“generic” (e.g., random, arbitrary, haphazard, etc.) arrays, since thetarget nucleic acid(s) are unknown, perfect match and mismatch probescannot be a priori determined, designed, or selected. In this instance,the probes can be provided as pairs where each pair of probes differs inone or more pre-selected nucleotides. Thus, while it is not known apriori which of the probes in the pair is the perfect match, it is knownthat when one probe specifically hybridizes to a particular targetsequence, the other probe of the pair will act as a mismatch control forthat target sequence. It will be appreciated that the perfect match andmismatch probes need not be provided as pairs, but may be provided aslarger collections (e.g., 3, 4, 5, or more) of probes that differ fromeach other in particular preselected nucleotides. While the mismatch(s)may be located anywhere in the mismatch probe, terminal mismatches areless desirable as a terminal mismatch is less likely to preventhybridization of the target sequence. In a particularly preferredembodiment, the mismatch is located at or near the center of the probesuch that the mismatch is most likely to destabilize the duplex with thetarget sequence under the test hybridization conditions. In aparticularly preferred embodiment, perfect matches differ from mismatchcontrols in a single centrally-located nucleotide.

It will be understood by one of skill in the art that control of thecharacteristics of the solution such as the stringency are important inusing the present invention to measure the binding characteristics of aanalyte-probe pair, or the concentration of a nucleic acid analyte(target nucleic acid). A variety of hybridization conditions may be usedin the present invention, including high, moderate and low stringencyconditions; see for example Maniatis et al., Molecular Cloning: ALaboratory Manual, 2d Edition, 1989, and Short Protocols in MolecularBiology, ed. Ausubel, et al, hereby incorporated by reference. Stringentconditions are sequence-dependent and will be different in differentcircumstances. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, “Overview of principlesof hybridization and the strategy of nucleic acid assays” (1993). Insome embodiments, highly stringent conditions are used. In otherembodiments, less stringent hybridization condition; for example,moderate or low stringency conditions may be used, as known in the art;see Maniatis and Ausubel, supra, and Tijssen, supra. The hybridizationconditions may also vary when a non-ionic backbone, i.e. PNA is used, asis known in the art.

Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences tend to hybridize specificallyat higher temperatures. Generally, stringent conditions can be selectedto be about 5.degree. C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength, pH, and nucleic acidconcentration) at which 50% of the probes complementary to the targetsequence hybridize to the target sequence at equilibrium. (As the targetanalyte sequences are generally present in excess, at T_(m), 50% of theprobes are occupied at equilibrium). Typically, stringent conditionswill be those in which the salt concentration is at least about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

In some embodiments, the probe and or the analyte may comprise anantibody. As used herein, the term “antibody” refers to animmunoglobulin molecule or a fragment of an immunoglobulin moleculehaving the ability to specifically bind to a particular molecule,referred to as an antigen. The antibody may be an anti-receptor antibodyspecific for the receptor used in the assay. Thus, the antibody may becapable of specifically binding the receptor as the antigen. Antibodiesand methods for their manufacture are well known in the art ofimmunology. The antibody may be produced, for example, by hybridoma celllines, by immunization to elicit a polyclonal antibody response, or byrecombinant host cells that have been transformed with a recombinant DNAexpression vector that encodes the antibody. Antibodies include but arenot limited to immunoglobulin molecules of any isotype (IgA, IgG, IgE,IgD, IgM), and active fragments including Fab, Fab′, F(ab′)₂, Facb, Fv,ScFv, Fd, V_(H) and V_(L). Antibodies include but are not limited tosingle chain antibodies, chimeric antibodies, mutants, fusion proteins,humanized antibodies and any other modified configuration of animmunoglobulin molecule that comprises an antigen recognition site ofthe required specificity.

The preparation of antibodies including antibody fragments and othermodified forms is described, for example, in “Immunochemistry inPractice,” Johnstone and Thorpe, Eds., Blackwell Science, Cambridge,Mass., 1996; “Antibody Engineering,” 2.sup.nd edition, C. Borrebaeck,Ed., Oxford University Press, New York, 1995; “Immunoassay”, E. P.Diamandis and T. K. Christopoulos, Eds., Academic Press, Inc., SanDiego, 1996; “Handbook of Experimental Immunology,” Herzenberg et al.,Eds, Blackwell Science, Cambridge, Mass., 1996; and “Current Protocolsin Molecular Biology” F. M. Ausubel et al., Eds., Greene Pub. Associatesand Wiley Interscience, 1987, the disclosures of which are incorporatedherein. A wide variety of antibodies also are available commercially.

In some embodiments, the probe and or the analyte may comprise twoproteins. Protein-protein interactions can enable two or more proteinsto associate. A large number of non-covalent bonds can form between theproteins when two protein surfaces are precisely matched, and thesebonds account for the specificity of recognition. Protein-proteininteractions are involved, for example, in the assembly of enzymesubunits; of multiprotein enzymatic complexes, or of molecular machines;in enzyme-substrate reactions; in antigen-antibody reactions; in formingthe supramolecular structures of ribosomes, filaments, and viruses; intransport; and in the interaction of receptors on a cell with growthfactors and hormones. Products of oncogenes can give rise to neoplastictransformation through protein-protein interactions. For example, someoncogenes encode protein kinases whose enzymatic activity on cellulartarget proteins leads to the cancerous state. Another example of aprotein-protein interaction occurs when a virus infects a cell byrecognizing a polypeptide receptor on the surface, and this interactionhas been used to design antiviral agents. In some cases, protein-proteininteractions can be dependent on protein modifications. For example,histone proteins can be modified at different positions with differentchemical tags (e.g. phosphorylation, or methylation), and themodifications themselves be required or involved in the recognition byother proteins (e.g chromatin remodeling and associated proteins).

Binding Kinetics

One aspect of the current invention is the use of the measurement of thebinding kinetics to characterize binding of multiple probes and analytesin solution. The term “binding kinetics” as used herein refers to therate at which the binding of the analyte to the probe occurs in abinding reaction. The term “binding reaction” as used herein describesthe reaction between probes and analytes. In some cases, bindingreaction refers to the concurrent binding reactions of multiple analytesand probes, and in other cases, the term binding reaction refers to thereaction between a single probe with a single analyte. The meaning willbe clear from the context of use. The kinetic measurements can beexpressed as the amount of analyte bound to the probe as a function oftime. The binding kinetics can provide information about thecharacteristics of the probe-analyte binding such as the strength ofbinding, the concentration of analyte, the competitive binding of ananalyte, the density of the probes, or the existence and amount ofcross-hybridization.

In order to determine binding kinetics, the signal at multiple timepoints must be determined. The signal at at least two time points isrequired. In most cases, more than two time points will be desired inorder to improve the quality of the kinetic information. In someembodiments the signal at, 2-10, 10-50, 50-100, 100-200, 200-400,400-800, 800-1600, 1600-3200, 3200-6400, 6400-13000, or higher than13,000 time points will be measured. One of ordinary skill in the artcan determine the effective number of points for a given embodiment. Forexample, where few points are obtained, the quality of information aboutthe binding event can be low, but where the number of data points isvery high, the data quality may be high, but the handling, storage, andmanipulation of the data can be cumbersome.

The frequency at which the signal is measured will depend on thekinetics of the binding reaction or reactions that are being monitored.As the frequency of measurements gets lower, the time betweenmeasurements gets longer. One way to characterize a binding reaction isto refer to the time at which half of the analyte will be bound(t_(1/2)). The binding reactions of the invention can have a (t_(1/2))from on the order of milliseconds to on the order of hours, thus thefrequency of measurements can vary by a wide range. The time betweenmeasurements will generally not be even over the time of the bindingreaction. In some embodiments, a short time between of measurements willbe made at the onset of the reaction, and the time between measurementswill be longer toward the end of the reaction. One advantage of thepresent invention is the ability to measure a wide range of bindingrates. A high initial frequency of measurements allows thecharacterization of fast binding reactions which may have higherbinding, and lower frequency of measurements allows the characterizationof slower binding reactions. For example, points can initially bemeasured at a time between points on the order of a microsecond, thenafter about a millisecond, points can be measured at a time betweenpoints on the order of a millisecond, then after about a second, timepoints can be measured at a time between points on the order of asecond. Any function can be used to ramp the change in measurementfrequency with time. In some cases, as described below, changes in thereaction conditions, such as stringency or temperature changes will bemade during a reaction, after which it may be desirable to change thefrequency of measurements to measure the rates of reaction which will bechanged by the change in reaction condition.

In some embodiments, a probe will have substantially no analyte bound toit at the beginning of the binding reaction, then the probe will beexposed to a solution containing the analyte, and the analyte will beginto bind, with more analyte bound to the probe with time. In some cases,the reaction will reach saturation, the point at which all of theanalyte that is going to bind has bound. Generally, saturation willoccur when a reaction has reached steady state. At steady state, in agiven time period, just as many analytes are released as new analytesare bound (the on rate and off rate are equal). In some cases, with verystrong binding, where the off-rate for the analyte is essentially zero,saturation will occur when substantially all of the analyte that canbind to the probe will have bound, has bound. Thus, while it isadvantageous to measure a change in signal with time that can becorrelated with binding kinetics, the measurement of a signal that doesnot change with time also provides information in the context of areal-time experiment, and can also be useful in the present invention.For example, in some cases the absence of a change in the signal willindicate the level of saturation. In other cases the absence of a changein signal can indicate that the rate of the reaction is very slow withrespect to the change in time measured. It is thus a beneficial aspectof this invention to measure binding event in real time both wheresignals change with time and where the signals do not change with time.

One aspect of the methods of the present invention is the measurement ofconcentration of an analyte from the measurement of binding kinetics.Since analyte binding rate can be concentration-dependant, we canestimate the analyte abundance in the sample solution using bindingrates.

In some embodiments, the concentration of analyte can be determined byequations relating to the kinetics of the hybridization process. Forexample, suppose that the number of probes at a particular spot on thearray prior to any hybridization is given by P₀. The probability of aspecific target binding to the probe site is given by

Prob(binding)=k·Prob(target and probe in close proximity·Prob(probe isfree),  (1)

where k≦1 depends of the bonds between the probe and the target andessentially a function of temperature, incubation conditions, and probedensity. Here, the first probability is proportional to the number oftarget molecules available whereas the second probability is

$\begin{matrix}{{{{Probe}\left( {{probe}\mspace{14mu} {is}\mspace{14mu} {free}} \right)} = \frac{P(t)}{P_{0}}},} & (2)\end{matrix}$

where P(t) is the number of available probes at time t, i.e., those arenot yet bound to any target. If we thus denote the forward and backwardstarget/probe binding reaction rates by K₊ and K, respectively, we maywrite the following differential equation for the available probeconcentration P(t):

$\begin{matrix}{\frac{{P(t)}}{t} = {{{- \frac{K_{+}}{P_{0}}}{P(t)}\left( {C - P_{0} - {P(t)}} \right)} + {K_{-}\left( {P_{0} - {P(t)}} \right)}}} & (3)\end{matrix}$

where C is the original target quantity in the solution so thatC−(P₀−P(t)) represents the available target density at time t. The aboveis a Riccati differential equation that can be solved in closed form.However, instead of doing so, we can note that for small values oft wehave P(t)≅P₀, so that the differential equation becomes

$\begin{matrix}{\frac{{P(t)}}{t} = {{- \frac{K_{+}}{P_{0}}}{P(t)}{C.}}} & (4)\end{matrix}$

This a first-order linear differential equation with time constantτ=P₀/K₊C. Accordingly, the target density can be determined from thereaction rate (or time constant) of P(t). In other words, using manysample measurements of P(t) at different times and fitting them to thecurve

$\begin{matrix}{{P(t)} = {P_{0}{\exp \left( {{- \frac{K_{+}}{P_{0}}}C} \right)}t}} & (5)\end{matrix}$

allows us to estimate the target quantity C. In this case, the reactionrate (or inverse of the time constant) is proportional to the targetconcentration and inversely proportional to the probe density, somethingthat has been observed in experiments.

One can also attempt to estimate C from the steady-state value of P(t),i.e., P_(∞). This can be found by setting dP(t)/dt=0 in the originalRiccati equation which leads to a quadratic equation for P_(∞). In somesimple cases, the solution to this quadratic equation can beconsiderably simplified.

When the target concentration is low: In this case, we can assume P₀>>C,so that we obtain

P _(∞) =P ₀ −C,  (6)

i.e., the reduction in available probes is equal to the targetconcentration.

When the target concentration is high: In this case, we can assume thatP₀<<C, so that we obtain

$\begin{matrix}{P_{\infty} = {\frac{K_{-}}{K_{+}} \cdot {\frac{P_{0}^{2}}{C}.}}} & (6)\end{matrix}$

In this case, the number of remaining probes is inversely proportional othe target concentration. This corresponds to probe saturation, whichgenerally is not as good a method of determining C as determining Cbased on the reaction rate near the beginning of the reaction.

One aspect of the present invention is the determination of the bindingof analyte to probe by measuring the rate near the beginning of thereaction. In addition to providing a more reliable estimate of C,measurement near the beginning of the reaction can shorten the time thatis required to measure analyte binding over the time required formeasuring binding from saturation. In some embodiments of the invention,the binding is measured during the time for less than about the first0.1, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 18, 20, 25, 30, 40, 50, 60,70, 80, or 90 percent of the analyte to bind as compared to the amountof analyte bound at saturation. In some embodiments, the bindingkinetics are determined in a time for less than about the first 20% ofthe analyte to bind. In some embodiments, the binding kinetics aredetermined in a time for less than about the first 1-2% of the analyteto bind.

Changing Conditions During the Binding Experiment

One aspect of the methods of the present invention is a step of changingthe conditions during the binding experiment. In conventionalmicroarrays where only the end-point is determined, only a single set ofbinding conditions can be tested. In the methods of the presentinvention, the binding conditions can be changed in order to exploremultiple sets of binding conditions during the same binding experiment.The condition which is changed can be, for example, any condition thataffects the rate of binding of analyte to probe. The condition which ischanged can be, for example, temperature, pH, stringency, analyteconcentration, ionic strength, an electric field, or the addition of acompetitive binding compound.

In some embodiments, the condition that is changed changes the rate ofbinding or hybridization. When measuring the binding of multipleanalytes to probes in the same binding medium, as in the presentinvention, the kinetics of binding can vary widely for differentanalyte-probe combinations. The binding rate conditions can be varied,for example, by changing the temperature, concentration, ionic strength,pH, or by applying an electric potential. The binding rates for thedifferent analytes can in some cases vary by many orders of magnitude,making it difficult and time consuming to measure the binding of all theanalytes in one binding experiment. This ability to change the rateconditions can be used to improve the measurement of binding formultiple analytes that bind at different rates, for example byperforming the initial part of the experiment under slower rateconditions, such that rapidly binding analytes can be readily measured,then raising the rate conditions such that more slowly binding analytescan be readily measured. This method of changing the binding rate duringthe binding experiment can also be used for better characterization of asingle analyte or single set of analytes in solution, for instance,using binding rate conditions to measure the initial portion of thebinding kinetics, then increasing the binding rate conditions to measurethe later portion of the binding kinetics for a single analyte, forexample, to establish the level of saturation. It will be understood bythose of skill in the art that this method of changing the rateconditions can result in both improved quality of measurements, such asthe measurement of analyte concentration, and/or in a savings of time.With the present method, for example by measuring the kinetics ofbinding, then changing the conditions to increase the rate of binding ofweaker binding species, the time of the binding experiment can bereduced by greater than about 10%, 20%, 50%, 75%, or by a factor of 2,4, 8, 10, 50, 100, 1000 or greater than 1000 over the times needed toobtain the same quality information using end-point binding methods.

In some embodiments, the condition that is changed is the stringency. Asdescribed above, the stringency can be changed by many factors includingtemperature, ionic strength, and the addition of compounds such asformamide. In some embodiments of the present invention, the medium isat one stringency at the beginning of the binding reaction, and at alater point the stringency of the medium is changed. This method can beused where different analytes or sets of analytes have differenthybridization characteristics, for example, allowing the measurement ofthe binding of one set of analytes with a high stringency, then allowingthe measurement of another set of analytes at a lower stringency in thesame medium as part of the same binding experiment. This method can alsobe used for the characterization of binding for a single analyte by, forinstance, measuring binding at high stringency at an initial portion ofthe binding reaction, then lowering the stringency and measuring a laterportion of the binding reaction. The ability to change stringency canalso be used to create conditions where a bound analyte becomes unbound,allowing, for instance, the measurement of the kinetics of binding atone stringency, followed by the measurement of release of the analyteinto solution upon raising the stringency. This method also allows thebinding of an analyte to a probe to be measured multiple times, forexample, by measuring the kinetics of binding of the analyte under oneset of stringency conditions, changing the stringency to release theanalyte, for instance, by raising the stringency, then measuring thekinetics of binding of the analyte a subsequent time by changing thestringency conditions again, for example by lowering the stringency.Thus the ability to change the stringency during the binding reactionallows for the measurement of any number of binding and unbindingreactions with the same set of probes and analytes.

In some embodiments of the method of the present invention, an electricpotential is applied during the binding reaction to the fluid volume toelectrically change the stringency of the medium. In some embodiments,the system will provide an electrical stimulus to the capturing regionusing an electrode structure which is placed in proximity of thecapturing region. If the analyte is an electro-active species and/orion, the electrical stimulus can apply an electrostatic force of theanalyte. In some embodiments the electrical potential is direct current(DC). In some embodiments, the electric potential is time-varying. Insome embodiments the electric potential has both DC and time varyingaspects. Their amplitude of the applied potential can be between 1 mV to10V, but typically between 10 mV to 100 mV. The frequency oftime-varying signal is between 1 Hz to 1000 MHz, but typically between100 Hz to 100 kHz.

In some embodiments, the change in conditions is the addition of acompetitive binding agent. For example, initially, a sample can beintroduced which contains analyte that binds to a particular probe. Thebinding of that analyte can be monitored as described herein. Then, atany point during the binding of that analyte, an analyte that competesfor the binding of that analyte to the probe can be added. The rate ofdisplacement of the analyte by the competitive binding agent can then bemeasured, providing more information to characterize the binding ofanalyte to probe.

Detection of Signals

For the methods of the present invention, a signal is detected that canbe correlated with the binding of analytes to the plurality of probes.The type of signals appropriate for the invention is any signal that canbe amount of analyte bound to the plurality of probes. Appropriatesignals include, for example, electrical, electrochemical, magnetic,mechanical, acoustic, or electromagnetic (light) signals. Examples ofelectrical signals useful in the present invention that can becorrelated with analyte binding are capacitance and/or impedance. Forexample, analytes labeled with metals or metal clusters can change thecapacitance and/or the impedance of a surface in contact with a fluid,allowing the amount of analyte bound to the probe on the surface to bedetermined. The electrical measurement can be made at any frequencyincluding DC, 0-10 Hz, 10-100 Hz, 100-1000 Hz, 1 KHz-10 KHz, 10 KHz-100KHz, 100 KHz-1 MHz, 1 MHz-10 MHZ, 10 MHz-100 MHz, 100 MHz-1 GHz, orabove 1 GHz. In some embodiments, impedance spectroscopy can be usedwhich obtains impedance versus frequency for any range of frequencieswithin the range of frequencies described above. Examples ofelectrochemical signals useful in the present invention that can becorrelated with analyte binding include amperometric and voltammetricmeasurements, and/or measurements that involve the oxidation orreduction of redox species. For example, the analyte can be labeled witha compound which undergoes an oxidation or reduction reaction at a knownredox potential, and the oxidative or reductive current can becorrelated with the amount of analyte bound to surface probes. Examplesof mechanical signals include the use of microelectomechanical (MEMS)devices. For example, the binding of analyte to probe on the surface ofa small surface feature, such as a cantilever, can change the mass ofthe surface feature, the vibration frequency of which can then becorrelated with the amount of analyte bound to the probe. Generally, thehigher the mass, the lower the vibration frequency. Examples of acousticsignals include surface acoustic wave (SAW), and surface plasmonresonance signals. A surface acoustic wave (SAW) is an acoustic wavetraveling along the surface of a material having some elasticity, withamplitude that typically decays exponentially with the depth of thesubstrate. The binding of labeled or unlabeled analyte to probe on asurface can change the SAW characteristics, e.g. amplitude, frequency ina manner that can be correlated with the amount of analyte bound to aprobe. Surface plasmon resonance relies on surface plasmons, also knownas surface plasmon polaritons, which are surface electromagnetic wavesthat propagate parallel, usually along a metal/dielectric interface.Since the wave is on the boundary of the metal and the external medium(water for example), these oscillations are very sensitive to any changeof this boundary, such as the adsorption of molecules to the metalsurface. The binding of labeled or unlabeled analyte to a probe attachedto the surface can change the frequency of the resonant surface plasmonin a manner that can be correlated with the amount of analyte bound tothe probes.

Particularly useful signals for the methods of the present invention areelectromagnetic (light) signals. Examples of optical signals useful inthe present invention are signals from fluorescence, luminescence, andabsorption. As used herein, the terms “electromagnetic” or“electromagnetic wave” and “light” are used interchangeably.Electromagnetic waves of any frequency and wavelength that can becorrelated to the amount of analyte bound to probe on the surface can beused in the present invention including gamma rays, x-rays, ultravioletradiation, visible radiation, infrared radiation, and microwaves. Whilesome embodiments are described with reference to visible (optical)light, the descriptions are not meant to limit the embodiments to thoseparticular electromagnetic frequencies.

For the methods of the present invention it is desired that the signalchanges upon the binding of the analyte to the probe in a manner thatcorrelates with the amount of analyte bound. In some cases, the changein signal will be a change in intensity of the signal. In someembodiments, the signal intensity will increase as more analyte is boundto probe. In some embodiments, the signal intensity will decrease asmore analyte is bound to probe. In some embodiments, the change insignal is not a change in intensity, but can be any other change in thesignal that can be correlated with analyte binding to probe. Forexample, the change in signal upon binding of the probe can be a changein the frequency of the signal. In some embodiments, the signalfrequency will increase as more analyte is bound to probe. In someembodiments, the signal frequency will decrease as more analyte is boundto the probe.

The signal that is measured is generally the signal in the region of thesolid surface. In some embodiments, signal from moieties attached to thesurface is used as the signal that can be correlated with the amount ofanalyte bound to the probe. In some embodiments signal from the solutionis used as the signal that can be correlated with the amount of analytebound to the probe.

In some embodiments of the methods of the present invention, labels areattached to the analytes and/or the probes. Any label can be used on theanalyte or probe which can be useful in the correlation of signal withthe amount of analyte bound to the probe. It would be understood bythose of skill in the art that the type of label with is used on theanalyte and/or probe will depend on the type of signal which is beingused, for example, as described above, a dense label for a mechanicalsignal, or a redox active label for a voltammetric measurement.

In some embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to the buildup of label at the surface asmore analyte is bound to the probes on the surface. For example, wherethe analyte has a fluorescent label, as more analyte binds, theintensity of the fluorescent signal can increase in a manner that can becorrelated with the amount of analyte bound to probe on the surface. Insome embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to the release of label from the surface.For example, where the probe has a fluorescent label and the label isreleased into solution upon the binding of the analyte to the probe, thefluorescent intensity at the surface will decrease as more analyte isbound and more fluorescent label is released.

In some embodiments, the signal that can be correlated to the amount ofanalyte bound to probe is due to a change in the signal from label onthe surface upon binding of the analyte to the probe. For example, wherea fluorescent label is on the surface, and the analyte is labeled with acompound capable of changing the fluorescent signal of the surfacefluorescent label upon binding of the analyte with the probe, the changein signal can be correlated with the amount of analyte bound to probe.In some embodiments, the analyte is labeled with a quencher, and thedecrease in intensity from the surface fluorescent label due toquenching is correlated to the increased amount of analyte bound toprobe. In some embodiments, the analyte is labeled with a fluorescentcompound which can undergo energy transfer with the fluorescent label onthe surface such that the increase in fluorescence from the analytefluorescent label and/or the decrease in fluorescence from the surfacefluorescent label can be correlated with the amount of analyte bound toprobe. In some embodiments the surface fluorescent label is bounddirectly, e.g. covalently to the probe. In some embodiments, the surfacefluorescent label is bound to the surface, is not bound to the probe,but is in sufficient proximity that the binding of the analyte to theprobe produces a change in signal from the surface fluorescent labelthat can be correlated with the amount of analyte bound to probe.

In some embodiments, the analyte is unlabeled, and the bindingcharacteristics and or concentration of the analyte is determined bycompetitive binding with another labeled species, which competes withthe analyte for biding to a probe. For example, where we have a solutionwith an analyte, A, whose concentration we want to determine, and wehave a competitive binding species, B, whose binding characteristicswith probe and whose concentration are known, then using the presentinvention, we can use, for example, an array of probes on a surface todetermine the concentration of A by determining the amount ofcompetitive binding of B to a probe. For example, the probe is attachedto a surface that is fluorescently labeled, and B is labeled with aquencher such that the level of quenching of the surface fluorescencecan be correlated with the amount of B bound to the probe. The rate ofbinding of B to the probe is measured in real time, and theconcentration of A is determined by knowing the characteristics of A asa competitive binder. In some embodiments, the amount of the competitivebinding species does not need to be known beforehand. For instance, thekinetics of binding of be can be measured in the fluid volume, then theconditions can be changed, (e.g. increasing the stringency) such that Bis released from the probe, then the analyte A is added, and the bindingof B under competition with A is measured. This example illustrates anadvantage of the being able to change the conditions of the mediumduring one experiment. In some cases, A and B can be the same species,where B is labeled, and the amount of B is known, and the amount of Acan be determined by the kinetics of the binding of B. In some cases, Aand B are not the same species, but compete for binding with a probe:This competitive binding real-time assay can be done with all types ofmolecular species described herein including nucleic acids, antibodies,enzymes, binding proteins, carbohydrates and lipids.

Electromagnetic Signals—Optical Methods

The use of optical detection provides a variety of useful ways ofimplementing the methods of the present invention. Optical methodsinclude, without limitation, absorption, luminescence, and fluorescence.

Some embodiments of the invention involve measuring light absorption,for example by dyes. Dyes can absorb light within a given wavelengthrange allowing for the measurement of concentration of molecules thatcarry that dye. In the present invention, dyes can be used as labels,either on the analyte or on the probe. The amount of dye can becorrelated with the amount of analyte bound to the surface in order todetermine binding kinetics. Dyes can be, for example, small organic ororganometallic compounds that can be, for example, covalently bound tothe analyte to label the analyte. Dyes which absorb in the ultraviolet,visible, infrared, and which absorb outside these ranges can be used inthe present invention. Methods such as attenuated total reflectance(ATR), for example for infrared, can be used to increase the sensitivityof the surface measurement.

Some embodiments of the invention involve measuring light generated byluminescence. Luminescence broadly includes chemiluminescence,bioluminescence, phosphorescence, and fluorescence. In some embodiments,chemiluminescence, wherein photons of light are created by a chemicalreaction such as oxidation, can be used. Chemiluminescent species usefulin the invention include, without limitation, luminol, cyalume, TMAE(tetrakis(dimethylamino)ethylene), oxalyl chloride, pyrogallol(1,2,3-trihydroxibenzene), lucigenin. In some embodiments,bioluminescence is used. Where the luminescence is bioluminescence,creation of the excited state derives from an enzyme catalyzed reaction.Bioluminescence derives from the capacity of living organisms to emitvisible light through a variety of chemiluminescent reaction systems.Bioluminescence generally include three major components: a luciferin, aluciferase and molecular oxygen. However other components may also berequired in some reactions, including cations (Ca⁺⁺ and Mg⁺⁺) andcofactors (ATP, NAD(P)H). Luciferases are enzymes that catalyze theoxidation of a substrate, luciferin, and produce an unstableintermediate. Light is emitted when the unstable intermediate decays toits ground state, generating oxyluciferin. Any of the differentunrelated types of luciferin can be used herein including those fromphyla which use a luciferin, known as coelenterazine, which contains aring formed by three amino acids (2 tyrosines, and a phenylalanine).Photoproteins from animals such as jellyfish can be used where the“photoprotein” of the luciferin/luciferase system emits light uponcalcium binding. Other bioluminescent systems as described in U.S.Patent Application 2007/0065818, and including bioluminescence resonanceenergy transfer (BRET) as described in U.S. Patent Application2007/0077609 can be used in the present invention.

Fluorescent Systems

A useful embodiment of the present invention involves the use offluorescence. As used herein, fluorescence refers to the process whereina molecule relaxes to its ground state from an electronically excitedstate by emission of a photon. As used herein, the term fluorescencealso encompasses phosphorescence. For fluorescence, a molecule ispromoted to an electronically excited state by generally by theabsorption of ultraviolet, visible, or near infrared radiation. Theexcited molecule then decays back to the ground state, or to alower-lying excited electronic state, by emission of light. An advantageof fluorescence for the methods of the invention is its highsensitivity. Fluorimetry may achieve limits of detection several ordersof magnitude lower than for absorption. Limits of detection of 10⁻¹⁰ Mor lower are possible for intensely fluorescent molecules; in favorablecases under stringently controlled conditions, the ultimate limit ofdetection (a single molecule) may be reached.

A wide variety of fluorescent molecules can be utilized in the presentinvention including small molecules, fluorescent proteins and quantumdots. Useful fluorescent molecules (fluorophores) include, but are notlimited to: 1,5 IAEDANS; 1,8-ANS; 4-Methylumbelliferone;5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM);5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA);5-FAM (5-Carboxyfluorescein); 5-HAT (Hydroxy Tryptamine); 5-HydroxyTryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 5-TAMRA(5-Carboxytetramethylrhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD);7-Hydroxy-4-methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine; ABQ;Acid Fuchsin; ACMA (9-Amino-6-chloro-2-methoxyacridine); AcridineOrange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin FeulgenSITSA; Aequorin (Photoprotein); AFPs—AutoFluorescent Protein—(QuantumBiotechnologies); Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™;Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™;Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™;Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S;AMCA (Aminomethylcoumarin); AMCA-X; Aminoactinomycin D; Aminocoumarin;Aminomethylcoumarin (AMCA); Anilin Blue; Anthrocyl stearate; APC(Allophycocyanin); APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G;Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine;ATTO-TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine;BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH);Berberine Sulphate; Beta Lactamase; Bimane; Bisbenzamide; Bisbenzimide(Hoechst); bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™-3;Bodipy 492/515; Bodipy 493/503; Bodipy 500/510; Bodipy 505/515; Bodipy530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589;Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676;Bodipy FI; Bodipy FL ATP; Bodipy FI-Ceramide; Bodipy R6G SE; Bodipy TMR;Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP;Bodipy TR-X SE; BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC;BTC-5N; Calcein; Calcein Blue; Calcium Crimson™; Calcium Green; CalciumGreen-1 Ca.sup.2+Dye; Calcium Green-2 Ca.sup.2+; Calcium Green-5NCa.sup.2+; Calcium Green-C18 Ca.sup.2+; Calcium Orange; CalcofluorWhite; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow;Catecholamine; CCF2 (GeneBlazer); CFDA; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coumarin Phalloidin; C-phycocyanine; CPMMethylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8;Cy5.5™; Cy5™; Cy7™; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl;Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansylfluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3′ DCFDA; DCFH(Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123);Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di-16-ASP);Dichlorodihydrofluorescein Diacetate (DCFH); DiD—Lipophilic Tracer; DiD(DiIC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DiIC18(3));Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DiIC18(7)); DM-NERF (high pH);DNP; Dopamine; DTAF; DY-630-NHS; DY-635-NHS; ELF 97; Eosin; Erythrosin;Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1);Euchrysin; EukoLight; Europium (III) chloride; EYFP; Fast Blue; FDA;Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC;Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate;Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX;FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2;Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF;Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer (CCF2); Gloxalic Acid;Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold);Hydroxytryptamine; Indo-1, high calcium; Indo-1, low calcium;Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf;JC-1; JO-JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751(RNA); Leucophor PAF; Leucophor SF; LeucophorWS; Lissamine Rhodamine;Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1;Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso TrackerGreen; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue;LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red(Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; MagnesiumGreen; Magnesium Orange; Malachite Green; Marina Blue; Maxilon BrilliantFlavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin;Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; MitotrackerRed; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH);Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine;Nile Red; Nitrobenzoxadidole; Noradrenaline; Nuclear Fast Red; NuclearYellow; Nylosan Brilliant lavin E8G; Oregon Green; Oregon Green 488-X;Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514;Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed [Red 613]; Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-PRO-3; Primuline;Procion Yellow; Propidium lodid (PL); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Red 613[PE-TexasRed]; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110;Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green;Rhodamine Phallicidine; Rhodamine Phalloidine; Rhodamine Red; RhodamineWT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); S65A; S65C; S65L;S65T; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G;Sevron Brilliant Red B; Sevron Orange; Sevron Yellow L; SITS; SITS(Primuline); SITS (Stilbene Isothiosulphonic Acid); SNAFL calcein;SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua;SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ(6-methoxy-N-(3-sulfopropyl)quinolinium); Stilbene; Sulphorhodamine Bcan C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange;Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange;Thioflavin 5; Thioflavin S; Thioflavin TCN; Thiolyte; Thiozole Orange;Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5;TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; TruRed; Ultralite; UranineB; Uvitex SFC; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H;Y66W; YO-PRO-1; YO-PRO-3; YOYO-1;YOYO-3, Sybr Green, Thiazole orange(interchelating dyes), or combinations thereof.

Some embodiments of the present invention include the Alexa Fluor dyeseries (from Molecular Probes/Invitrogen) which cover a broad spectrumand match the principal output wavelengths of common excitation sourcessuch as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546,555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750. Someembodiments of the present invention include the Cy Dye fluorophoreseries (GE Healthcare), also covering a wide spectrum such as Cy3, Cy3B,Cy3.5, Cy5, Cy5.5, Cy7. Some embodiments of the present inventioninclude the Oyster dye fluorophores (Denovo Biolabels) such asOyster-500, -550, -556, 645, 650, 656. Some embodiments of the presentinvention include the DY-Labels series (Dyomics), for example, withmaxima of absorption that range from 418 nm (DY-415) to 844 nm (DY-831)such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556,-560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647,-648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700,-701, -730, -731, -732, -734, -750, -751, -752, -776, -780, -781, -782,-831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL. Some embodimentsof the present invention include the ATTO fluorescent labels (ATTO-TECGmbH) such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590,594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740.Some embodiments of the present invention include CAL Fluor and Quasardyes (Biosearch Technologies) such as CAL Fluor Gold 540, CAL FluorOrange 560, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL FluorRed 635, Quasar 670. Some embodiments of the present invention includequantum dots such as the EviTags (Evident Technologies) or quantum dotsof the Qdot series (Invitrogen) such as the Qdot 525, Qdot565, Qdot585,Qdot605, Qdot655, Qdot705, Qdot 800. Some embodiments of the presentinvention include fluorescein, rhodamine, and/or phycoerythrin.

FRET and Quenching

In some embodiments of the invention, fluorescence resonance energytransfer is used to produce a signal that can be correlated with thebinding of the analyte to the probe. FRET arises from the properties ofcertain fluorophores. In FRET, energy is passed non-radiatively over adistance of about 1-10 nanometers between a donor molecule, which is afluorophore, and an acceptor molecule. The donor absorbs a photon andtransfers this energy non-radiatively to the acceptor (Forster, 1949, Z.Naturforsch. A4: 321-327; Clegg, 1992, Methods Enzymol. 211: 353-388).When two fluorophores whose excitation and emission spectra overlap arein close proximity, excitation of one fluorophore will cause it to emitlight at wavelengths that are absorbed by and that stimulate the secondfluorophore, causing it in turn to fluoresce. The excited-state energyof the first (donor) fluorophore is transferred by a resonance induceddipole—dipole interaction to the neighboring second (acceptor)fluorophore. As a result, the excited state lifetime of the donormolecule is decreased and its fluorescence is quenched, while thefluorescence intensity of the acceptor molecule is enhanced anddepolarized. When the excited-state energy of the donor is transferredto a non-fluorophore acceptor, the fluorescence of the donor is quenchedwithout subsequent emission of fluorescence by the acceptor. In thiscase, the acceptor functions as a quencher.

Pairs of molecules that can engage in fluorescence resonance energytransfer (FRET) are termed FRET pairs. In order for energy transfer tooccur, the donor and acceptor molecules must typically be in closeproximity (up to 7 to 10 nanometers. The efficiency of energy transfercan falls off rapidly with the distance between the donor and acceptormolecules.

Molecules that can be used in FRET include the fluorophores describedabove, and includes fluorescein, 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), rhodamine,6-carboxyrhodamine (R6G), N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA), 6-carboxy-X-rhodamine (ROX), 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL), and 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS). Whether a fluorophore is a donor or an acceptor is definedby its excitation and emission spectra, and the fluorophore with whichit is paired. For example, FAM is most efficiently excited by light witha wavelength of 488 nm, and emits light with a spectrum of 500 to 650nm, and an emission maximum of 525 nm. FAM is a suitable donorfluorophore for use with JOE, TAMRA, and ROX (all of which have theirexcitation maximum at 514 nm).

In some embodiments of the methods of the present invention, theacceptor of the FRET pair is used to quench the fluorescence of thedonor. In some cases, the acceptor has little to no fluorescence. TheFRET acceptors that are useful for quenching are referred to asquenchers. Quenchers useful in the methods of the present inventioninclude, without limitation, Black Hole Quencher Dyes (BiosearchTechnologies such as BHQ-0, BHQ-1, BHQ-2, BHQ-3, BHQ-10; QSY Dyefluorescent quenchers (from Molecular Probes/Invitrogen) such as QSY7,QSY9, QSY21, QSY35, and other quenchers such as Dabcyl and Dabsyl; Cy5Qand Cy7Q and Dark Cyanine dyes (GE Healthcare), which can be used, forexample, in conjunction with donor fluors such as Cy3B, Cy3, or Cy5;DY-Quenchers (Dyomics), such as DYQ-660 and DYQ-661; and ATTOfluorescent quenchers (ATTO-TEC GmbH), such as ATTO 540Q, 580Q, 612Q.

In some embodiments of the methods of the invention, both the analytesand the probes have labels that are members of a FRET pair, and thelabels are attached such that when an analyte binds to a probe, FRETwill occur between the labels, resulting in a change in signal that canbe correlated with the binding of analyte to probe in real-time. Thechange in signal can be the decrease in the intensity of the donorand/or the increase in the intensity of the acceptor. The FRET pair canbe chosen such that emission wavelength of the donor fluorophore is farenough from the emission wavelength of the acceptor fluorophore, thatthe signals can be independently measured. This allows the measurementof both the decrease in signal from the donor and the increase in signalfrom the acceptor at the same time, which can result in improvements inthe quality of the measurement of binding. In some cases, the probe willhave a label that is the donor of the donor-acceptor pair. In somecases, the analyte will have a label that is the donor of the donoracceptor pair.

In some embodiments of the methods of the invention, the analyte willhave a fluorescent label that is a member of a FRET pair, and the othermember of the FRET pair will be attached to the surface, wherein themember of the FRET pair attached to the surface is not covalently linkedto the probe. In some cases, the analyte will have a label that is thedonor of the donor-acceptor pair. In some cases, the analyte will have alabel that is the acceptor of the donor acceptor pair. In someembodiments, the member of the FRET pair that is attached to the surfaceis attached to an oligonucleotide which is attached to the surface (asurface-bound label). The oligonucleotide that is labeled with the FRETpair can be a nucleotide sequence that does not have a sequenceanticipated to specifically bind to an analyte. The use of asurface-bound label allows for the labeling of multiple areas of anarray without having to label each specific binding probe. This cansimplify the production of the array and reduce costs. We have foundthat even though the surface-bound FRET pairs are not covalently boundto the probe, they can be sensitive to the binding of the analytelabeled with the other member of the FRET pair in a manner that allowsthe change in signal to be correlated with the amount of analyte boundto probe.

In some embodiments of the methods of the present invention, the analyteis labeled with a quencher, and the probe is labeled with a donorfluorophore. The analyte is labeled with the quencher such that whenanalyte binds with the probe, the fluorescence from the fluorescentlabel on the probe is quenched. Thus, the signal, measured in real-time,can be correlated with the amount of binding of the analyte and theprobe, allowing for the measurement of the kinetics of the binding. Insome embodiments of the methods of the present invention, the analyte islabeled with a quencher, and the probe is labeled with a donorfluorophore, that is not covalently attached to it. The quencher islabeled such that when analyte binds with the probe, the fluorescencefrom the fluorescent label on the probe is quenched. Thus, the signal,measured in real-time, can be correlated with the amount of binding ofthe analyte and the probe, allowing for the measurement of the kineticsof the binding.

In some embodiments of the methods of the present invention, the analyteis labeled with a quencher, and the surface is labeled with a donorfluorophore wherein the donor fluorophore is not covalently linked tothe probe (e.g. with a surface bound fluorescent label). The quencher islabeled such that when analyte binds with the probe, the fluorescencefrom the fluorescent label on the surface is quenched. Thus, the signal,measured in real-time, can be correlated with the amount of binding ofthe analyte and the probe, allowing for the measurement of the kineticsof the binding.

Where the probe is labeled with a fluorophore, one aspect of theinvention is the use of an image of the fluorescently labeled probe onthe surface obtained before binding has occurred in order to effectivelyestablish a baseline signal for the state where no binding of analyte toprobe has occurred. In conventional arrays, in which unlabeled probe istreated with labeled analyte, and the signal is measured afterhybridization and washing, it can be difficult to know exactly how muchprobe is actually on the array in the region of interest. Thus,differences in array manufacture can affect the quality of the data. Inthe present invention, where the probe is labeled with fluorophore, theimage of the labeled probe on the surface provides a measurement of theamount of probe actually on the surface, increasing the quality andreliability of the binding measurement.

One exemplary embodiment of the method of the invention is illustratedin FIGS. 5 and 6. FIG. 5B shows a top view of a 4 by 4 microarray thathas 16 independently addressable spots, each spot having bound DNAprobes, wherein the probes are labeled with fluorescent label. FIG. 5Cshows a close up view of one of the spots illustrating the attachedprobe of sequence (A), each probe having a fluorescent label. FIG. 5Dshows the close up view of a second spot with attached probes ofsequence (B), each probe having a fluorescent label. FIG. 5A shows aside view of the array, showing that the array is in contact with thehybridization solution. FIG. 5 represents a time at which no analyte isbound to probe on the array.

FIG. 6 illustrates the same array as in FIG. 5 after hybridization forsome time with target analytes (targets) having a quencher attached.FIGS. 6A and 6B shows a side view and top view of the array, still incontact with the hybridization solution. The different spots on thearray in FIG. 6B have different light intensities, indicating that thereis a different amount of binding of analyte at each spot, and thereforea different amount of fluorescence from the spots. FIG. 6C shows a closeup view illustrating that a small amount of target (A) has specificallybound (hybridized) to probe (A) resulting in quenching of each moleculeof probe to which analyte is bound. FIG. 6D illustrates that a largeramount of analyte (B) has specifically bound (hybridized) to probe (B),resulting in a higher level of quenching than observed for spot (A). Thesignal from each of the spots on the array can be measured at varioustime points during the binding reaction between analytes and probes,while the solution containing the analyte is in contact with the solidsurface of the microarray, allowing a real-time measurement of theamount of analyte-probe binding, and allowing the measurement of bindingkinetics at each spot.

Measuring Cross-Hybridization

One aspect of the methods of the present invention is the step ofperforming an algorithm on real-time binding data to determinecross-hybridization for multiple probes on a substrate. One embodimentinvolves improving the quality of analyte-probe binding measurements bydetermining and correcting for cross-hybridization.

In its early stages, the probe-target binding kinetics can be describedby a simple first-order differential equation whose time-domain solutionis given by an exponential function; the rate of decay of theexponential function is determined by the binding reaction rate.Non-specific binding can have an adverse effect on the accuracy ofmicroarray platforms, especially if the amount of the non-specifictarget is high relative to the specific target. One reason is that thespecific and non-specific targets will compete for the same probe, andeven though the probability of non-specific binding is much lower it mayhave an effect if the amount of the non-specific target is much higher.Fortunately, specific and non-specific bindings have different reactionrates, by virtue of the fact that the binding probabilities and targetamounts are different; therefore, if both specific and non-specific(i.e., interfering) targets bind to a probe on a microarray or aparallel affinity-based biosensor, the signal measured by a RT-μArraysystem can be represented by a sum of exponentials,

μ(t)=Σα_(j)exp(β_(j) t),  (1)

where Σα_(j) gives the initial intensity of the probe spots, the β_(j)are the rates of decay for the specific (j=1) and non-specific (j=2,3, .. . ) bindings, and where the α_(j) themselves depend on the reactionrates and target densities. In certain embodiments of the invention, weemploy the so-called Prony method or one of its modifications. Thismethod essentially represents the signal μ(t) in terms of thecoefficients of the original differential equation. These coefficientsare computed by finding eigenvalues of an appropriate covariance matrix.In other embodiments of the invention, algebraic characterization usingsingular value decomposition of the correlation matrix of the data isemployed. In yet another embodiment of the invention,information-theoretic or Bayesian techniques can be used to detect apresence of the species that bind non-specifically, estimate the numberof such species, and quantify their amounts.

The Basic Algorithm

The hybridization process in general satisfies a nonlinear differentialequation. To see this, let p(t) denote the number of available probemolecules at a given spot at time t, and let n(t) denote the number ofanalytes in the solution that are specific to this spot. Thus, if theprobability that an analyte be in close proximity to a probe molecule isP_(near), the probability that it binds to the probe once it is near ina unit interval of time is P_(h), and the probability that an analytebound to a probe molecule is released in a unit interval of time isP_(r), then we may write

p(t+δ)−p(t)=δ(p ₀ −p(t))P _(r) −δp(t)n(t)P _(near) P _(h),  (1)

where p₀ denotes the initial number of probe molecules on this probespot of the array. If we denote the total number of analyte molecules byN, then it is clear that n(t)=N−p₀+p(t). Letting δ→0, therefore gives

$\begin{matrix}{\frac{{q(t)}}{t} = {{\left( {p_{0} - {p(t)}} \right)P_{r}} - {{p(t)}\left( {N - p_{0} + {p(t)}} \right)P_{near}{P_{h}.}}}} & (2)\end{matrix}$

Upon rearranging terms, we have

$\begin{matrix}{{\frac{{p(t)}}{t} = {{p_{0}P_{r}} - {{p(t)}\left\lbrack {P_{r} + {\left( {N - p_{0}} \right)P_{near}P_{h}}} \right\rbrack} - {{p^{2}(t)}P_{near}P_{h}}}},} & (3)\end{matrix}$

which is the nonlinear equation we were seeking. This is a Riccatiequation and can be solved in closed form (however, we shall not do sohere). In any event, it is clear that by looking at the trajectory ofp(t) one can glean information about the parameters N, p₀, P_(h), P_(r).For example, the steady-state of p(t), i.e., p_(∞)=p(∞), satisfies thequadratic equation

P _(near) =P _(h) p _(∞) ² +[P _(r)+(N−p ₀)P _(near) P _(h) ]p _(∞) −p ₀P _(r)=0  (4)

and so measuring it, gives us information about the parameters ofinterest.

Determining the Analyte Concentration

The equations are often more instructive when written in terms of thenumber of probe molecules that have bound to analytes, i.e.,q(t)=p₀−p(t). In this case we may write

$\begin{matrix}{{{- \frac{{q(t)}}{t}} = {{{q(t)}P_{r}} - {\left( {p_{0} - {q(t)}} \right)\left( {N - {q(t)}} \right)P_{near}P_{h}}}},} & (5)\end{matrix}$

which upon a rearrangement of terms becomes

$\begin{matrix}{\frac{{q(t)}}{t} = {{{Np}_{0}P_{near}P_{h}} - {\left\lbrack {P_{r} + {\left( {N + p_{0}} \right)P_{near}P_{h}}} \right\rbrack {q(t)}} + {P_{near}P_{h}{{q^{2}(t)}.}}}} & (6)\end{matrix}$

In the early phase of the hybridization process, i.e., when q(t) is verysmall, we may ignore the quadratic term in the differential equation andwrite

$\begin{matrix}{\frac{{q(t)}}{t} \approx {{{Np}_{0}P_{near}P_{h}} - {\left\lbrack {P_{r} + {\left( {N + p_{0}} \right)P_{near}P_{h}}} \right\rbrack {{q(t)}.}}}} & (7)\end{matrix}$

This has the solution

$\begin{matrix}{{q(t)} \approx {\frac{{Np}_{0}P_{near}P_{h}}{P_{r} + {\left( {N + p_{0}} \right)P_{near}P_{h}}}{\left( {1 - {\exp \left( {- {t\left\lbrack {P_{r} + {\left( {N + p_{0}} \right)P_{near}P_{h}}} \right\rbrack}} \right)}} \right).}}} & (8)\end{matrix}$

The above formula has several different ramifications; first the slopeof increase of q(t), equivalently, the slope of decay of p(t), is givenby

$\begin{matrix}{{\left. \frac{{q(t)}}{t} \right|_{t = 0} = {{Np}_{0}P_{near}P_{h}}},} & (9)\end{matrix}$

which is proportional to the number of analytes in the solution, N.

Second, the reaction rate also has information on the number ofanalytes. In fact, the reaction rate is simply the coefficient of t inthe exponential function for q(t), which is readily seen to be

reaction_rate=P _(r)+(N+p ₀)P _(near) P _(h).  (10)

Though this is not quite a linear relationship for N, it can still beused to estimate the number of analytes.

Of course, one can estimate N jointly from both the initial slope andthe reaction rate. In particular, it is often true that P_(r) is verysmall and can be ignored compared to other terms. Therefore we shalltake

reaction_rate=(N+p ₀)P _(near) P _(h)  (11)

Determining and Suppressing Cross-Hybridization

The other advantage of looking at the reaction rate is that it allowsfor one to deal with cross-hybridization and to suppress its effect. Asmentioned earlier, in the early phase of the hybridization process, thenumber of available probe molecules, or equivalently the light intensityof a probe spot, decays exponentially with time. For example

C _(k)exp(−r _(k) t),  (12)

where C_(k) is a constant and r_(k) is the reaction rate that can bedetermined from the parameters of the experiment (probability ofhybridization, number of analytes, number of probe molecules, etc.). Inparticular, as seen from (11), r_(k) is linear in the number of targetanalytes, N_(k), say.

If, in addition to hybridization of the target of interest, a number ofother targets cross-hybridize to the same probe spot, the lightintensity of the probe spot will decay as the sum of severalexponentials, i.e.,

$\begin{matrix}{{{I(t)} = {\sum\limits_{k = 0}^{K - 1}{C_{k}\; {\exp \left( {{- r_{k}}t} \right)}}}},} & (13)\end{matrix}$

where k=0 corresponds to the desired target and k=1, . . . , K−1corresponds to the K−1 cross-hybridizing analytes. The whole point isthat the reaction rates for the different analytes differ (due todifferent numbers of analytes, binding probabilities, etc.) so that ifwe can estimate the reaction rates from (13), we should be able todetermine the number of molecules for each different analyte.

The RT-uArray system samples the signal (i.e., the light intensity) ofthe probe spots at certain time intervals (multiples of Δ, say) and thusobtains the sequence

$\begin{matrix}{y_{n} = {{{I\left( {n\; \Delta} \right)} + {v\left( {n\; \Delta} \right)}} = {{\sum\limits_{k = 0}^{K - 1}{C_{k}\; {\exp \left( {{- n}\; \Delta \; r_{k}} \right)}}} + {v\left( {n\; \Delta} \right)}}}} & (14)\end{matrix}$

for n=0, 1, . . . T−1, where T is the total number of samples and v(t)represents the measurement noise. Defining u_(k)=exp(−Δr_(k)), we maywrite

$\begin{matrix}{y_{n} = {{\sum\limits_{k = 0}^{K - 1}\; {C_{k}u_{k}^{n}}} + {v_{n}.}}} & (15)\end{matrix}$

The goal is to (i) determine the value of K (i.e., how many analytes arebinding to the probe spot), (ii) to estimate the values of the pairs{C_(k), u_(k)} for all k=1, . . . , K−1 and (iii) to determine thenumber of each analyte N_(k) (recall from (9) that C_(k) is proportionalto the number of analytes and that from (11) the reaction rate is linearin N_(k)).

The problem of determining the number of exponential signals in noisymeasurements, and estimating the individual rates, is a classical one insignal processing and is generally referred to as system identification.(There are a multitude of books and papers on this subject.) The basicidea is that, when y_(n) is the sum of K exponentials, it satisfies a Kth order recurrence equation

y _(n) +h ₁ y _(n-1) + . . . . +h _(K-1) y _(n-K+1) +h _(K) y_(n-K)=0.  (16)

Furthermore, the u_(k) are the roots of the polynomial

H(z)=z ^(K) +h ₁ z ^(Kn-1) + . . . +h _(K-1) z+h _(k).  (17)

In practice, since one observes a noisy signal, one first uses themeasurements to form the so-called Hankel matrix

$\begin{matrix}\begin{pmatrix}y_{T/2} & y_{{T/2} - 1} & \cdots & y_{1} & y_{0} \\y_{{T/2} + 1} & y_{T/2} & \cdots & y_{2} & y_{1} \\\vdots & \vdots & \ddots & \vdots & \vdots \\y_{T} & y_{T - 1} & \cdots & y_{{T/2} + 1} & y_{T/2}\end{pmatrix} & (18)\end{matrix}$

When y_(n) is the sum of K exponentials, the above Hankel matrix hasrank K, i.e., only K nonzero eigenvalues. When y_(n) is noisy, thestandard practice is to compute the singular values of the Hankel matrixand estimate K as being the number of significant singular values.

Once K has been determined, one forms the (T−K+1)×(K+1) Hankel matrix

$\begin{matrix}\begin{pmatrix}y_{K} & y_{K - 1} & \cdots & y_{1} & y_{0} \\y_{K + 1} & y_{K} & \cdots & y_{2} & y_{1} \\\vdots & \vdots & \ddots & \vdots & \vdots \\y_{T} & y_{T - 1} & \cdots & y_{T - K + 1} & y_{T - K}\end{pmatrix} & (19)\end{matrix}$

and then identifies the vector (1 h₁ . . . h_(k))^(t) with the smallestright singular vector of (19).

As mentioned earlier, the roots of H(z) are the desired u_(k), fromwhich we determine the rates r_(k) and thereby the amounts of targetspresent. While the algorithm outlined above can be used, a variety ofdifferent techniques to find the u_(k), including, but not limited to,total least squares, ESPRIT, Prony's method, may be used.

In addition to the algorithm described above, other algorithmicsolutions can be used, including the methods overviewed in (Petersson etal., Applied Mathematics and Computation, vol. 126, no. 1, February2002, pp. 31-61). The methods described above provide the ability toquantify the amounts of the species that bind whether specifically ornon-specifically.

Arrays

One aspect of the invention is an array that has a solid surface with aplurality of probes attached to it, where the array can be used for thereal-time measurement of binding of analyte to the plurality of probes.

The arrays of the present invention comprise probes attached to a solidsubstrate. The solid substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides,semiconductor integrated chips, etc. The solid substrate is preferablyflat but may take on alternative surface configurations. For example,the solid substrate may contain raised or depressed regions on whichsynthesis or deposition takes place. In some embodiments, the solidsubstrate will be chosen to provide appropriate light-absorbingcharacteristics. For example, the substrate may be a polymerizedLangmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂,SiN₄, modified silicon, or any one of a variety of gels or polymers suchas (poly)tetrafluoroethylene, (poly)vinylidendifluoride, polystyrene,polycarbonate, or combinations thereof.

The substrate can be a homogeneous solid and/or unmoving mass muchlarger than the capturing probe where the capturing probes are confinedand/or immobilized within a certain distance of it. The mass of thesubstrate is generally at least 100 times larger than capturing probesmass. In certain embodiments, the surface of the substrate is planarwith roughness of 0.1 nm to 100 nm, but typically between 1 nm to 10 nm.In other embodiments the substrate can be a porous surface withroughness of larger than 100 nm. In other embodiments, the surface ofthe substrate can be non-planar. Examples of non-planar substrates arespherical magnetic beads, spherical glass beads, and solid metal and/orsemiconductor and/or dielectric particles.

In some embodiments the substrate is optically clear, allowing light tobe transmitted through the substrate, and allowing excitation and ordetection to occur from light passing through the substrate. In someembodiments the substrate is opaque. In some embodiments, the substrateis reflective, allowing for light to pass through the surface layercontaining probes and reflect back to a detector.

In some embodiments, glass slides are used to prepare biochips. Thesubstrates (such as films or membranes) can also be made of silica,silicon, plastic, metal, metal-alloy, anopore, polymeric, and nylon. Thesurfaces of substrates can be treated with a layer of chemicals prior toattaching probes to enhance the binding or to inhibit non-specificbinding during use. For example, glass slides can be coated withself-assembled monolayer (SAM) coatings, such as coatings of asaminoalkyl silanes, or of polymeric materials, such as acrylamide andproteins. A variety of commercially available slides can be used. Someexamples of such slides include, but are not limited to, 3D-link®(Surmodics), EZ-Rays® (Mosaic Technologies), Fastslides® (Schleicher andSchuell), Superaldehyde®, and Superamine® (CEL Technologies).

Probes can be attached covalently to the solid surface of the substrate(but non-covalent attachment methods can also be used). In oneembodiment, similar substrate, coating, and attachment chemistries areused for all three—UniScreen™, ProScreen™, NuScreen™—devices. In anotherembodiment, different chemistries are applied.

A number of different chemical surface modifiers can be added tosubstrates to attach the probes to the substrates. Examples of chemicalsurface modifiers include N-hydroxy succinimide (NHS) groups, amines,aldehydes, epoxides, carboxyl groups, hydroxyl groups, hydrazides,hydrophobic groups, membranes, maleimides, biotin, streptavidin, thiolgroups, nickel chelates, photoreactive groups, boron groups, thioesters,cysteines, disulfide groups, alkyl and acyl halide groups, glutathiones,maltoses, azides, phosphates, and phosphines. Glass slides with suchchemically modified surfaces are commercially available for a number ofmodifications. These can easily be prepared for the rest, using standardmethods (Microarray Biochip Technologies, Mark Schena, Editor, March2000, Biotechniques Books).

In one embodiment, substrate surfaces reactive towards amines are used.An advantage of this reaction is that it is fast, with no toxicby-products. Examples of such surfaces include NHS-esters, aldehyde,epoxide, acyl halide, and thio-ester. Most proteins, peptides,glycopeptides, etc. have free amine groups, which will react with suchsurfaces to link them covalently to these surfaces. Nucleic acid probeswith internal or terminal amine groups can also be synthesized, and arecommercially available (e.g., from IDT or Operon). Thus, nucleic acidscan be bound (e.g., covalently or non-covalently) to surfaces usingsimilar chemistries.

The substrate surfaces need not be reactive towards amines, but manysubstrate surfaces can be easily converted into amine-reactivesubstrates with coatings. Examples of coatings include amine coatings(which can be reacted with bis-NHS cross-linkers and other reagents),thiol coatings (which can be reacted with maleimide-NHS cross-linkers,etc.), gold coatings (which can be reacted with NHS-thiol cross linkers,etc.), streptavidin coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, biotin-NHS cross-linkers,etc.), and BSA coatings (which can be reacted with bis-NHScross-linkers, maleimide-NHS cross-linkers, etc.). Alternatively, theprobes, rather than the substrate, can be reacted with specific chemicalmodifiers to make them reactive to the respective surfaces.

A number of other multi-functional cross-linking agents can be used toconvert the chemical reactivity of one kind of surface to another. Thesegroups can be bifunctional, tri-functional, tetra-functional, and so on.They can also be homo-functional or hetero-functional. An example of abi-functional cross-linker is X-Y-Z, where X and Z are two reactivegroups, and Y is a connecting linker. Further, if X and Z are the samegroup, such as NHS-esters, the resulting cross-linker, NHS-Y-NHS, is ahomo-bi-functional cross-linker and would connect an amine surface withan amine-group containing molecule. If X is NHS-ester and Z is amaleimide group, the resulting cross-linker, NHS-Y-maleimide, is ahetero-bi-functional cross-linker and would link an amine surface (or athiol surface) with a thio-group (or amino-group) containing probe.Cross-linkers with a number of different functional groups are widelyavailable. Examples of such functional groups include NHS-esters,thio-esters, alkyl halides, acyl halides (e.g., iodoacetamide), thiols,amines, cysteines, histidines, di-sulfides, maleimide, cis-diols,boronic acid, hydroxamic acid, azides, hydrazines, phosphines,photoreactive groups (e.g., anthraquinone, benzophenone), acrylamide(e.g., acrydite), affinity groups (e.g., biotin, streptavidin, maltose,maltose binding protein, glutathione, glutathione-S-transferase),aldehydes, ketones, carboxylic acids, phosphates, hydrophobic groups(e.g., phenyl, cholesterol), etc. Such cross-linkers can be reacted withthe surface or with the probes or with both, in order to conjugate aprobe to a surface.

Other alternatives include thiol reactive surfaces such as acrydite,maleimide, acyl halide and thio-ester surfaces. Such surfaces cancovalently link proteins, peptides, glycopeptides, etc., via a (usuallypresent) thiol group. Nucleic acid probes containing pendantthiol-groups can also be easily synthesized.

Alternatively, one can modify glass surfaces with molecules such aspolyethylene glycol (PEG), e.g. PEGs of mixed lengths

Other surface modification alternatives (such as photo-crosslinkablesurfaces and thermally cross-linkable surfaces) are known to thoseskilled in the art. Some technologies are commercially available, suchas those from Mosiac Technologies (Waltham, Mass.), Exiqon™ (Vedbaek,Denmark), Schleicher and Schuell (Keene, N.H.), Surmodics™ (St. Paul,Minn.), Xenopore™ (Hawthorne, N.J.), Pamgene (Netherlands), Eppendorf(Germany), Prolinx (Bothell, Wash.), Spectral Genomics (Houston, Tex.),and Combimatrix™ (Bothell, Wash.).

Surfaces other than glass are also suitable for such devices. Forexample, metallic surfaces, such as gold, silicon, copper, titanium, andaluminum, metal oxides, such as silicon oxide, titanium oxide, and ironoxide, and plastics, such as polystyrene, and polyethylene, zeolites,and other materials can also be used. The devices can also be preparedon LED (Light Emitting Diode) and OLED (Organic Light Emitting Diode)surfaces. An array of LEDs or OLEDs can be used at the base of a probearray. An advantage of such systems is that they provide easyoptoelectronic means of result readout. In some cases, the results canbe read-out using a naked eye.

Probes can be deposited onto the substrates, e.g., onto a modifiedsurface, using either contact-mode printing methods using solid pins,quill-pins, ink-jet systems, ring-and-pin systems, etc. (see, e.g., U.S.Pat. Nos. 6,083,763 and 6,110,426) or non-contact printing methods(using piezoelectric, bubble-jet, syringe, electro-kinetic, mechanical,or acoustic methods. Devices to deposit and distribute probes ontosubstrate surfaces are produced by, e.g., Packard Instruments. There aremany other methods known in the art. Preferred devices for depositing,e.g., spotting, probes onto substrates include solid pins or quill pins(Telechem/Biorobotics).

The arrays of the present invention can also be three-dimensional arrayssuch as porous arrays. Such as devices consisting of one or more porousgel-bound probes in an array or an array of arrays format. A device canhave one or more such structures and the structures can be of anygeometric shape and form. The structures can also be verticallystraight, angled, or twisted. Thus, each device denotes a (multiplexed)reaction site. The device can be used to perform reactionssimultaneously or sequentially. Any of the known substrates andchemistries can be used to create such a device. For example, glass,silica, silicon wafers, plastic, metals; and metal alloys can all beused as the solid support (see. e.g., Stillman B A, Tonkinson J L,Scleicher and Schuell; Biotechniques, 29(3), 630-635, 2000; Rehmna et.al; Mosaic Technologies Inc., Nucleic Acids Research, 27(2), 649-655,1999). In other embodiments, the intermediate species can be immobilizedto the substrate using mechanical and/or electrostatic and/or andmagnetic forces. Examples are magnetic beads with magnetic fields andglass beads with electrostatic fields. Bead based methods are described,for example in Gunderson et al., Genome Research, 870-877, 2004; Michaelet al., Anal. Chem. 70, 1242-1248, 1998; Han et al., Nat. Biotechnol,19, 631-635, 2001; and Lockhart et al., Nat. Biotechnol. 19, 1122-1123,2001.

In other embodiments, the microarrays are manufactured through thein-situ synthesis of the probes. This in-situ synthesis can be achievedusing phosphoramidite chemistry and/or combinatorial chemistry. In somecases, the deprotection steps are performed by photodeprotection (suchas the Maskless Array Synthesizer (MAS) technology, (NimbleGen; or thephotolithographic process, by Affymetrix). In other cases, deprotectioncan be achieved electrochemically (such as in the Combimatrixprocedure). Microarrays for the present invention can also bemanufactured by using the inkjet technology (Agilent).

For the arrays of the present invention, the plurality of probes may belocated in one addressable region and/or in multiple addressable regionson the solid substrate. In some embodiments the solid substrate hasabout 2, 3, 4, 5, 6, or 7-10, 10-50, 50-100, 100-500, 500-1,000,1,000-5,000, 5,000-10,000, 10,000-50,000, 50,000-100,000,100,000-500,000, 500,000-1,000,000 or over 1,000,000 addressable regionswith probes.

The spots may range in size from about 1 nm to 10 mm, in someembodiments from about 1 to 1000 micron and more in some embodimentsfrom about 5 to 100 micron. The density of the spots may also vary,where the density is generally at in some embodiments about 1 spot/cm²,in some embodiments at least about 100 spots/cm² and in otherembodiments at least about 400 spots/cm², where the density may be ashigh as 10⁶ spots/cm² or higher.

The shape of the spots can be square, round, oval or any other arbitraryshape.

One aspect of the invention is an array that comprises a solid substratehaving a surface and a plurality of different probes, wherein (a) thedifferent probes are immobilized to the surface at different addressablelocations, (b) the addressable locations comprise optical signalmoieties bound to the surface, (c) the optical signal moieties are notbound directly to the probes, and (d) the optical signal from theoptical signal moieties is capable of changing upon binding of ananalyte to the probes. For these arrays, the optical signal moiety, forexample, a fluorescent moeity is bound directly to the surface, but isnot covalently bound to a probe, and in these cases the probe need notbe labeled. The fluorescent moiety can be bound to the surface orsynthesized in-situ by an of the methods described above for probes. Thefluorescent moiety can be attached to an oligonucleotide that is not aprobe, for example, having a sequence that is not complementary totarget analytes in solution.

In one embodiment, a fluorescent moiety on the surface (surface-boundlabel) can be brought to the proximity of the probe viapost-probe-synthesis or post-probe-deposition methods.

In some embodiments, the label can be bound to the probe by non-covalentmeans, such as by hybridization. For example, in certain embodiments ofthe present invention, some or all of the probes on the microarray maycontain two different sequence segments: one segment that consists of asequence that is specific to the probe and specific for the detection ofa given target analyte, and another segment that is a sequence that iscommon to all or many of the probes on the microarray. These twosequence segments can be immediately adjacent to each other on theprobe, or separated by a linker. In this embodiment, the microarray isfirst hybridized with a (labeled oligonucleotide that is complementaryto the common sequence segment, thus resulting in a microarray in whichthe spots or features where the probes are located also now containfluorescent labels. These non-covalently bound labels can be bound tothe probe such that FRET and or quenching of the label occurs uponbinding of an analyte to the specific portion of the probe. This methodcan be advantageous, for instance by (1) lowering the cost ofmanufacturing microarrays that can be used in the real-time platformand/or (2) enabling the use of in-situ synthesized arrays in the realtime platform. The labeled oligonucleotide can be a locked nucleic acid(LNA) oligonucleotide. LNA oligonucleotides can be useful because theLNA modification can result in enhanced hybridization properties (forexample, diminishing the sequence length that is needed to achieve acertain Tm) (Jepsen et al., Oligonucleotides. 2004; 14(2):130-46).

Systems

One aspect of the invention is a system comprising: (a) a device with(i) a solid support having a surface and (ii) a plurality of differentprobes, wherein the different probes are immobilized to the surface; (b)a fluid volume comprising an analyte wherein the fluid volume is incontact with the solid support, and (c) a detector assembly comprisingmeans to detect signals measured at multiple time points from each of aplurality of spots on the microarray while the fluid volume is incontact with the solid support.

The fluid volume can be introduced and held in the system by any methodthat will maintain the fluid in contact with the solid support. In manycases the fluid is held in a chamber. In some embodiments the chamber isopen on one face, in other embodiments the chamber will mostly enclosethe fluid. In some embodiments, the chamber will have one or more portsfor introducing and/or removing material (usually fluids) from thechamber. In some embodiments one side of the chamber comprises the solidsubstrate on which the probes are attached. In some embodiments thechamber is integral to the solid substrate. In some embodiments, thechamber is a sub-assembly to which the solid substrate with probes canbe removably attached. In some embodiments, some or all of the fluidchamber is an integral part of the instrument that comprises thedetector. The chamber can be designed such that the signal that can becorrelated with analyte-probe binding can be detected by a detectoroutside of the chamber. For instance, all or a portion of the chambercan be transparent to light to allow light in or out of the chamber tofacilitate excitation and detection of fluorophores.

The detector assembly can comprise a single detector or an array ofdetectors or transducers. As used herein, the terms detector andtransducer are used interchangeably, and refer to a component that iscapable of detecting a signal that can be correlated with the amount ofanalyte-probe binding. Where the detector system is an array oftransducers, in some embodiments, the detector system is a fixed arrayof transducers, wherein one or more transducers in the transducer arraycorresponds to one independently addressable area of the array. In someembodiments, the detector or the array of transducers scans the arraysuch that a given detector or transducer element detects signals fromdifferent addressable areas of the array during a binding reaction.

In some embodiments the detector array is in contact with the solidsubstrate. In some embodiments, the detector is at a distance away fromthe substrate. Where the detector is a distance away from the substrate,in some embodiments, the detector or detector array is capable ofscanning the substrate in order to measure signal from multipleaddressable areas. In some embodiments, the detector is an opticaldetector which is optically coupled to the substrate. The detector canbe optically coupled to the substrate, for example with one or morelenses or waveguides.

FIG. 9 shows an example of a real-time microarray system where thedetection system comprises a sensor array in intimate proximity of thecapturing spots. In this embodiment, individual sensors detect thebinding events of a single capturing spots.

In some embodiments, the detector is optically coupled through spatiallyconfined excitation. This method is useful to optically couple thesubstrate to detector for a small region of substrate with probes. Thismethod generally requires only a single detector, since only one regioncan create signal at a time. The method can be used in scanning systems,and is applicable in assays which an excitation is required fordetection, such as fluorescence spectroscopy or surface plasmonresonance (SPR) methods.

In some embodiments, the detector is optically coupled through imagingusing focal plane detector arrays: In this method the signal generatedfrom the system is focused on a focal point detector array. Thisapproach useful for optical detection systems where signal focusing canbe carried out using lenses and other optical apparatus. Examples ofdetectors in these embodiments are complementary metal oxidesemiconductor (CMOS) and charge coupled device (CCD) image sensors.

In some embodiments, the detector is optically coupled through surfaceimaging: In this method the detectors are placed in intimate proximityof the capturing probes such that the signal generated from thecapturing region can only be observed by the dedicated detector. If amicroarray with multiple capturing spots is used, multiple detectors areused, each dedicated to an individual spot. This method can be used inelectrochemical-, optical-, and magnetic-based biosensors.

In some embodiments, the detector is optically coupled through surfaceimaging using signal couplers: In this method the detectors are notplace in proximity of the capturing spots, however a signal coupler isused to direct signal from the capturing region to a detector. Thismethod is generally used in optical detection systems where the signalcoupling elements is a plurality of optical waveguide. Examples ofsignal coupling elements include fiber optic cables, fiber opticbundles, fiber optic faceplates, and light pipes.

The detectors of the present invention must be capable of capturingsignal at multiple time points in real time, during the bindingreaction. In some embodiments the detector is capable of measuring atleast two signals in less than about 1 psec, 5 psec, 0.01 nsec, 0.05nsec, 0.1 nsec, 0.5 nsec, 1 nsec, 5 nsec, 0.01 μsec, 0.05 μsec, 0.1μsec, 0.5 μsec, 1 μsec, 5 μsec, 0.01 msec, 0.05 msec, 0.1 msec, 0.5msec, 1 msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec, 1 sec, 5 sec,10 sec, or 60 sec.

In some embodiments the detector detects the signal at the substrate. Insome embodiments the detector will detect the signal in the solution. Insome embodiments, the detector will detect signal in both the solutionand at the substrate.

In some embodiments the detector system is capable of detectingelectrical, electrochemical, magnetic, mechanical, acoustic, orelectromagnetic (light) signals.

Where the detector is capable of detecting optical signals, the detectorcan be, for example a photomultiplier tube (PMT), a CMOS sensor, or a(CCD) sensor. In some embodiments, the detector comprises a fiber-opticsensor.

In some embodiments, the system comprising the detector is capable ofsensitive fluorescent measurements including synchronous fluorimetry,polarized fluorescent measurements, laser induced fluorescence,fluorescence decay, and time resolved fluorescence.

In some embodiments, the system comprises a light source, for example,for excitation of fluorescence. The light source is generally opticallycoupled to the substrate, for example with one or more lenses orwaveguides. The light source can provide a single wavelength, e.g. alaser, or a band of wavelengths.

FIG. 7 shows a block diagram of the components of systems of the presentinvention. The system comprises of (i) reaction chamber which includesthe microarray substrate, probes, analytes, and solution, (ii) heatingand cooling modules and temperature sensor, (iii) temperaturecontroller, and (iv) detector which is connected to an analysis block,where the latter is a part of a computing system.

FIG. 8 shows an example of a real-time microarray system where real-timebinding of BHQ2 quencher-labeled cDNA molecules were detected using afluorescent laser-scanning microscope. The substrate in this example wasa transparent glass slides and the probes were 25 bp Cy5-labeledoligonucleotides. The light source (laser) and detector were bothlocated on the back of the substrate.

In some embodiments, the system comprises an instrument that has adetector assembly, and a computing system, where the instrument canaccept a sub-assembly. The sub assembly comprises a chamber that willhold the fluid volume and the solid substrate having a surface and aplurality of probes. The sub-assembly can be loaded into the instrumentin order to monitor the reaction during the binding event, and aftersaturation.

In some embodiments, the system comprises: an assay assembly comprisingmeans to engage a microarray and means to perform an assay on a surfaceof the microarray; and a detector assembly comprising means to detectsignals measured at multiple time points from each of a plurality ofspots on the microarray during the performance of the assay.

In some embodiments, the means to perform the assay comprise acompartment wherein the surface of the microarray comprises a floor ofthe compartment and means to deliver reagents and analytes into thecompartment. Any method can be used to seal the microarray to thecompartment including using adhesives and gaskets to seal the fluid. Anymethod can be used to deliver reagents and analytes including usingsyringes, pipettes, tubing, and capillaries.

In some embodiments, the system comprises a means of controlling thetemperature. Control of temperature can be important to allow control ofbinding reaction rates, e.g. by controlling stringency. The temperaturecan be controlled by controlling the temperature at any place within thesystem including controlling the temperature of the fluid or thetemperature of the solid substrate. Any means can be used forcontrolling the temperature including resistive heaters, Peltierdevices, infrared heaters, fluid or gas flow. The temperatures can bethe same or different for solution or substrate or different parts ofeach. Ideally the temperature is consistently controlled within thebinding region. In some embodiments the temperature is controlled towithin about 0.01, 0.05, 0.1, 0.5, or 1° C.

In some embodiments the system is capable of changing the temperatureduring the binding reaction. In some embodiments, the temperature can berapidly changed during the binding reaction. In some embodiments, thesystem is capable of changing the temperature at a rate of temperaturechange corresponding to a change of 1° C. in less than about 0.01 msec,0.1 msec, 0.5 msec, 1 msec, 5 msec, 10 msec, 50 msec, 100 msec, 0.5 sec,1 sec, 10 sec, or 60 sec.

In other embodiments the temperature is changed slowly, graduallyramping the temperature over the course of the binding reaction.

One exemplary embodiment of changing the temperature during the bindingreaction involves a change in temperature to change the bindingstringency and probability. Most bindings in affinity-based biosensorsare a strong function of temperature, thus by changing temperature wecan alter the stringency and observe the capturing the new capturingprocess with a new set of capturing probabilities.

In some embodiments, the system is capable of measuring temperature inone or multiple locations in the solution or on the solid substrate. Thetemperature can be measured by any means including by thermometer,thermocouple, or thermochromic shift.

In some embodiments the system comprises a feedback loop for tempcontrol wherein the measured temperature is used as an input to thesystem in order to more accurately control temperature.

In some embodiments, the system comprises an apparatus to add or removematerial from the fluid volume. In some embodiments, the system can addor remove a liquid from the fluid volume. In some embodiments, thesystem is capable of adding or removing material from the fluid volumein order to change the: concentration, pH, stringency, ionic strength,or to add or remove a competitive binding agent. In some embodiments,the system is capable of changing the volume of the fluid volume duringthe reaction.

One exemplary embodiment of adding material to the fluid volume duringthe binding reaction comprises the addition of incubation buffer. Theincubation buffer is the buffer in which the analytes are residing. Byadding the incubation buffer, the concentration of analytes in thesystem will decrease and therefore the binding probability and kineticof binding will both decrease. Furthermore, if the reaction has alreadyreached equilibrium, the addition of the buffer will cause the system tomove another equilibrium state in time.

Another exemplary embodiment of adding material to the fluid volumeduring the binding reaction is adding a competing binding species. Thecompeting species can be of the same nature of the analyte but ingeneral they are molecules which have affinity to capturing probes. ForDNA microarrays for example, the competing species can be synthesizedDNA oligo-nucleotides with partially or completely complementarysequence to the capturing probes. In immunoassays, the competing speciesare antigens.

In some embodiments the system comprises elements to apply an electricpotential to the fluid volume to electrically change the stringency ofthe medium. In some embodiments, the system will provide an electricalstimulus to the capturing region using an electrode structure which isplaced in proximity of the capturing region. If the analyte is anelectro-active species and/or ion, the electrical stimulus can apply anelectrostatic force of the analyte. In certain embodiments, thiselectrostatic force is adjusted to apply force on the bonds betweenanalyte and capturing probe. If the force is applied to detach themolecule, the affinity of the analyte-probe interaction is reduced andthus the stringency of the bond is evaluated. The electrical stimulus isgenerally a DC and/or time-varying electrical potentials. Theiramplitude can be between 1 mV to 10V, but typically between 10 mV to 100mV. The frequency of time-varying signal can be between 1 Hz to 1000MHz, in some embodiments, the frequency of the time-varying signal isbetween 100 Hz to 100 kHz. The use of electric potential to controlstringency is described in U.S. Pat. No. 6,048,690.

In some embodiments the system comprises a computing system foranalyzing the detected signals. In some embodiments, the system iscapable of transferring time point data sets to the computing systemwherein each time point data set corresponds to detected signal at atime point, and the computing system is capable of analyzing the timepoint data sets, in order to determine a property related to the analyteand probe. The methods of the current invention can, in some cases,generate more data, sometimes significantly more data than forconventional microarrays. Thus a computer system and software that canstore and manipulate the data (for instance, images taken at timepoints) can be essential components of the system. The data can beanalyzed in real-time, as the reaction unfolds, or may be stored forlater access.

The information corresponding to detected signal at each time point canbe single values such as signal amplitude, or can be more complexinformation, for instance, where each set of signal informationcorresponds to an image of a region containing signal intensity valuesat multiple places within an addressable location.

The property related to analyte and/or probe can be, for example,analyte concentration, binding strength, or competitive binding, andcross-hybridization.

In some embodiments the computing system uses the algorithms describedabove for determining concentration and/or cross-hybridization.

One aspect of the invention is software for use in characterizingbinding between analyte and probe. In one embodiment, the softwarecarries out the steps of i) accessing stored images taken at differenttime points, ii) performing image processing to determine the locationof the spots and convert the data to a collection of time series (onefor each spot) representing the temporal behavior of the signalintensity for each spot, and iii) for each spot on the array determiningwhether a reaction has happened (this is often done by comparing withcontrol spots on the array). Optionally, the software can perform thesteps of iv) determining whether the reaction at each spot involves thebinding of a single analyte or multiple analytes (if, for example,cross-hybridization is occurring), v) estimating the reaction ratesusing statistical system identification methods. Examples of statisticalsystem identification methods include methods such as Prony's method. Inthe case that step iv) is used, (multiple bindings per spot), thereaction rate of each binding is determined, and vi) using the reactionrates to estimate the unknown quantity of interest (analyteconcentration, binding strength, etc.) using, for example optimalBayesian methods.

In some embodiments, the system will have software for interfacing withthe instrument, for example allowing the user to display information inreal-time and allowing for user to interact with the reaction (i.e., addreagents, change the temperature, change the pH, dilution, etc.).

Uses

Where the probe and analyte are nucleic acids, the present inventionprovides methods of expression monitoring and generic differencescreening. The term expression monitoring is used to refer to thedetermination of levels of expression of particular, typicallypreselected, genes. The invention allows for many genes, e.g. 10, 100,1,000, 10,000, 100,000 or more genes to be analyzed at once. Nucleicacid samples are hybridized to the arrays and the resultinghybridization signal as a function of time provides an indication of thelevel of expression of each gene of interest. In some embodiments, thearray has a high degree of probe redundancy (multiple probes per gene)the expression monitoring methods provide accurate measurement and donot require comparison to a reference nucleic acid.

In another embodiment, this invention provides generic differencescreening methods, that identify differences in the abundance(concentration) of particular nucleic acids in two or more nucleic acidsamples. The generic difference screening methods involve hybridizingtwo or more nucleic acid samples to the same oligonucleotide array, orto different oligonucleotide arrays having the same oligonucleotideprobe composition, and optionally the same oligonucleotide spatialdistribution. The resulting hybridizations are then compared allowingdetermination which nucleic acids differ in abundance (concentration)between the two or more samples.

Where the concentrations of the nucleic acids comprising the samplesreflects transcription levels genes in a sample from which the nucleicacids are derived, the generic difference screening methods permitidentification of differences in transcription (and by implication inexpression) of the nucleic acids comprising the two or more samples. Thedifferentially (e.g., over- or under) expressed nucleic acids thusidentified can be used (e.g., as probes) to determine and/or isolatethose genes whose expression levels differs between the two or moresamples.

The expression monitoring and difference screening methods of thisinvention may be used in a wide variety of circumstances includingdetection of disease, identification of differential gene expressionbetween two samples (e.g., a pathological as compared to a healthysample), screening for compositions that upregulate or downregulate theexpression of particular genes, and so forth.

EXAMPLES Example 1

FIG. 10 shows the layout of a 6×6 DNA microarray. Three different DNAprobes (1, 2, and Control) with three different concentrations (2 μM, 10μM, and 20 μM) are spotted and immobilized on the surface asillustrated. The probes contain a single Cy3 fluorescent molecule at the5′ end. The DNA targets in this experiment contain a quencher molecule.The analyte binding in this system results in quenching of fluorescentmolecules in certain spots. FIG. 11 shows a few samples of the real-timemeasurements of the microarray experiment wherein the control targetsare added to the system. As illustrated in FIG. 11, the spots arequenched due to analyte binding.

FIGS. 12-15 each show data for 4 different spots with similaroligonucletide capturing probes. The target DNA analyte is introduced inthe system at time zero and quenching (reduction of signal) occurs onlywhen binding happens. For FIG. 12, the light intensity coefficient ofvariation was about 15%, however the estimated time constant rate fromreal-time measurements had only 4.4% variations. For FIG. 13 the lightintensity coefficient of variation was about 15%, however the estimatedtime constant rate from real-time measurements had only 2.1% variations.For FIG. 14 the light intensity coefficient of variation was about 22%,however the estimated time constant rate from real-time measurements hadonly 6% variations. For FIG. 15 the light intensity coefficient ofvariation was about 22%, however the estimated time constant rate fromreal-time measurements had only a 4.8% variation.

In FIG. 16, the signals measured during two real-time experimentswherein target 2 is applied to the microarray, first at 2 ng and then at0.2 ng, are shown. The measured light intensities at the correspondingprobe spots decay over time as the targets to the probes bind and thequenchers come in close proximity to the fluorescent labels attached tothe end of the probes. The rate of the decay, which can be estimated bya curve fitting technique, is proportional to the amount of the targetpresent. The time constant of the measured process is defined as theinverse of the rate of decay. The ratio of the time constants of the twoprocesses is 10, which is precisely the ratio of the amounts of targetsapplied in the two experiments.

Example 2

This example provides a derivation of an algorithm, and the use of thealgorithm to determine analyte concentration from a real-time bindingdata. The derivation proceeds as follows:

Assume that the hybridization process starts at t=0, and considerdiscrete time intervals of the length Δt. Consider the change in thenumber of bound target molecules during the time interval (iΔt,(i+1)Δt). We can write

n _(b)(i+1)−n _(b)(i)[n _(t) −n _(b)(i)]p _(b)(i)Δt−n _(b)(i)p_(r)(i)Δt,

where n_(t) denotes the total number of target molecules, n_(b)(i) andn_(b)(i+1) are the numbers of bound target molecules at t=iΔt andt=(i+1)Δt, respectively, and where p_(b)(i) and p_(r)(i) denote theprobabilities of a target molecule binding to and releasing from acapturing probe during the i^(th) time interval, respectively. Hence,

$\begin{matrix}{\frac{{n_{b}\left( {i + 1} \right)} - {n_{b}(i)}}{\Delta \; t} = {{\left\lbrack {n_{t} - {n_{b}(i)}} \right\rbrack {p_{b}(i)}} - {{n_{b}(i)}{{p_{r}(i)}.}}}} & (1)\end{matrix}$

It is reasonable to assume that the probability of the target releasedoes not change between time intervals, i.e., p_(r)(i)=p_(r), for all i.On the other hand, the probability of forming a target-probe pairdepends on the availability of the probes on the surface of the array.If we denote the number of probes in a spot by n_(p), then we can modelthis probability as

$\begin{matrix}{{{p_{b}(i)} = {{\left( {1 - \frac{n_{b}(i)}{n_{p}}} \right) - p_{b}} = {\frac{n_{p} - {n_{b}(i)}}{n_{p}}p_{b}}}},} & (2)\end{matrix}$

where p_(b) denotes the probability of forming a target-probe pairassuming an unlimited abundance of probes.

By combining (1) and (2) and letting Δt→0, we arrive to

$\begin{matrix}{\frac{n_{b}}{t} = {{{\left( {n_{t} - n_{b}} \right)\frac{n_{p} - n_{b}}{n_{p}}p_{p}} - {n_{b}p_{r}}} = {{n_{t}p_{b}} - {\left\lbrack {{\left( {1 + \frac{n_{t}}{n_{p}}} \right)p_{b}} + p_{r}} \right\rbrack n_{b}} + {\frac{p_{b}}{n_{p}}n_{b}^{2}}}}} & (3)\end{matrix}$

Note that in (3), only n_(b)=n_(b)(t), while all other quantities areconstant parameters, albeit unknown. Before proceeding any further, wewill find it useful to denote

$\begin{matrix}{{\alpha = {{\left( {1 + \frac{n_{t}}{n_{p}}} \right)p_{b}} + p_{r}}},{\beta = {n_{t}p_{b}}},{\gamma = {\frac{p_{b}}{n_{p}}.}}} & (4)\end{matrix}$

Clearly, from (4),

$\begin{matrix}{{p_{b} = \frac{\beta}{n_{t}}},{n_{p} = \frac{p_{b}}{\gamma}},{p_{r} = {\alpha - {\left( {1 + \frac{n_{t}}{n_{p}}} \right){p_{b}.}}}}} & \;\end{matrix}$

Using (4), we can write (3) as

$\begin{matrix}{{\frac{n_{b}}{t} = {{\beta - {\alpha \; n_{b}} + {\gamma \; n_{b}^{2}}} = {\gamma \; \left( {n_{b} - \lambda_{1}} \right)\left( {n_{b} - \lambda_{2}} \right)}}},} & (5)\end{matrix}$

where λ₁ and λ₂ are introduced for convenience and denote the roots of

β−αn _(b) +γn _(b) ²=0.

Note that γ=β/(λ₁λ₂). The solution to (5) is found as

${n_{b}(t)} = {\lambda_{1} + {\frac{\lambda_{1}\left( {\lambda_{1} - \lambda_{2}} \right)}{{\lambda_{2}e^{{\beta {({\frac{1}{\lambda_{1}} \cdot \frac{1}{\lambda_{2}}})}}^{t}}} - \lambda_{1}}.}}$

We should point out that (3) describes the change in the amount oftarget molecules, n_(b), captured by the probes in a single probe spotof the microarray. Similar equations, possibly with different values ofthe parameters n_(p), n_(t), p_(b), and p_(r), hold for other spots andother targets.

Estimating Parameters of the Model

The following is an outline of a procedure for estimation of theparameters. Ultimately, by observing the hybridization process, we wouldlike to obtain n_(t), n_(p), p_(b), and p_(r). However, we do not alwayshave direct access to n_(b)(t) in (6), but rather to y_(b)(t)=kn_(b)(t),where k denotes a transduction coefficient. In particular, we observe

$\begin{matrix}{{{y_{b}(t)} = {\lambda_{1}^{*} + \frac{\lambda_{1}^{*}\left( {\lambda_{1}^{*} - \lambda_{2}^{*}} \right)}{{\lambda_{2}^{*}e^{{\beta {({\frac{1}{\lambda_{1}^{*}} \cdot \frac{1}{\lambda_{2}^{*}}})}}^{t}}} - \lambda_{1}}}},} & (7)\end{matrix}$

where λ₁*=kλ₁, λ₂*=kλ₂, and β*=kβ.

For convenience, we also introduce

$\begin{matrix}{{\gamma^{*} = {\frac{\beta^{*}}{\lambda_{1}^{*}\lambda_{2}^{*}} = \frac{\gamma}{k}}},\; {\alpha^{*} = {{\gamma^{*}\left( {\lambda_{1}^{*} + \lambda_{2}^{*}} \right)} = {\alpha.}}}} & (8)\end{matrix}$

From (5), it follows that

$\begin{matrix}{\beta^{*} = \left. \frac{y_{b}}{t} \middle| {}_{t = 0}. \right.} & (9)\end{matrix}$

Assume, without a loss of generality, that λ₁* is the smaller and λ₂*the larger of the two, i.e., λ₁*=min(λ₁, λ₂), and λ₂*=max(λ₁,λ₂). From(7), we find the steady-state of y_(b)(t),

λ₁*=lim y _(b)(t),t→∞.  (10)

So, from (9) and (10) we can determine β* and λ₁*, two out of the threeparameters in (7). To find the remaining one, λ₂*, one needs to fit thecurve (7) to the experimental data.

Having determined β*, λ₁*, and λ₂*, we use (8) to obtain α* and γ*.Then, we should use (4) to obtain pb, pr, n_(p), and n_(t) from α*, β*,and γ*. However, (4) gives us only 3 equations while there are 4unknowns that need to be determined. Therefore, we need at least 2different experiments to find all of the desired parameters. Assume thatthe arrays and the conditions in the two experiments are the same exceptfor the target amounts applied. Denote the target amounts by n_(t), andn_(t); on the other hand, p_(b) and p_(r) remain the same in the twoexperiments. Let the first experiment yield α₁*, β₁*, and γ₁*, and thesecond one yield α₂*, β₂*, and γ₂* (we note that γ₁*=γ₂*). Then it canbe shown that

$\begin{matrix}{{p_{b} = \frac{{\beta_{1}^{*}\gamma_{1}^{*}} - {\beta_{2}^{*}\gamma_{2}^{*}}}{\alpha_{1}^{*} - \alpha_{2}^{*}}},{and}} & (11) \\{p_{r} = {\alpha_{1}^{*} - p_{b} - {\frac{\beta_{1}^{*}\gamma_{1}^{*}}{p_{b}}.}}} & (12)\end{matrix}$

Moreover,

$\begin{matrix}{{n_{p} = \frac{p_{b}}{k\; \gamma_{1}^{*}}},{and}} & (13) \\{{n_{t_{1}} = {\frac{\beta_{1}^{*}\gamma_{1}^{*}}{p_{b}^{2}}n_{p}}},\; {n_{t_{2}} = {\frac{\beta_{2}^{*}\gamma_{2}^{*}}{p_{*}^{b}}{n_{p}.}}}} & (14)\end{matrix}$

We note that quantities (13)-(14) are known within the transductioncoefficient k, where k=y_(b)(0)/n_(p). To find k and thus unambiguouslyquantify n_(p), n_(t1), and n_(t2), we need to perform a calibrationexperiment (i.e., an experiment with a known amount of targets n_(t)).

Experimental Example

Here we describe the experiments designed to test the validity of theproposed model and demonstrate the parameter estimation procedure. Tothis end, two DNA microarray experiments are performed. The custom8-by-9 arrays contain 25mer probes printed in 3 different probedensities. The targets are Ambion mRNA Spikes, applied to the arrayswith different concentrations. The concentrations used in the twoexperiments are 80 ng/50 μl and 16 ng/50 μl. The signal measured in thefirst experiment, where 80 ng of the target is applied to the array, isshown in FIG. 17. The smooth line shown in the same figure representsthe fit obtained according to (7). In the second experiment, 16 ng ofthe target is applied to the array. The measured signal, and thecorresponding fit obtained according to (7), are both shown in FIG. 18.

Applying (11)-(14), we obtain

p _(h)=1.9×10⁻³ ,p _(r)=2.99×10⁻⁵.

Furthermore, we find that

n _(t1) /n _(t2)=β*₁/β*₂=3.75  (15)

Note that the above ratio is relatively close to its true value,80/16=5. Finally, assuming that one of the experiments is used forcalibration, we find that the value of the transduction coefficient isk=4.1×10⁻⁴, and that the number of probe molecules in the observed probespots is n_(p)=1.6×10⁻¹¹.

Example 3

This example shows how the methods and systems of the present inventioncan be used for measurement of gene expression. Real-time microarraytechnology can measure, for example, expression level differences fordifferent cell types or tissues, distinct developmental stages,cancerous versus normal cells or tissues, treated versus untreated cellsor tissues, mutant versus wild-type cells, tissues or organisms.

The gene expression profiles of inflorescences of the Arabidopsisthaliana floral homeotic mutants apetala1, apetala2, apetala3,pistillata, and agamous can be compared with that of wild-type plants.By combining the data sets from the individual mutant/wild typecomparisons, it is possible to identify a large number of genes thatare, within flowers, predicted to be specifically or at leastpredominantly expressed in one type of floral organ. For each sample,floral buds from approximately 50 plants are collected, and RNA isisolated from 100 mg of tissue with the RNeasy RNA Isolation Kit(Qiagen). To prepare labeled target material from those samples, an invitro transcription amplification method followed byaminoallyl-UTP-mediated labeling is used. In brief, first and secondstrand cDNA is synthesized from 3 μg of total RNA using a polyA-primerwith a T7 promoter sequence. Then in vitro transcription is performedusing the Megascript T7 kit (Amb ion), in the presence of aminoallyl-UTPand of a reduced amount of UTP, to incorporate the modified aaUTP intothe aRNA during the transcription process. Finally, dye molecules (Cy3Mono-Reactive Dye, Cy5 Mono-Reactive Dye, Amersham) are coupled to theamplified RNA and the dye-labeled RNA is fragmented beforehybridization. These and similar protocols are well established in themicroarray field and are well known to those skilled in the art, andcommercial kits are available for cDNA synthesis, in vitro transcriptionamplification, and amino-allyl labeling, such as the Amino AllylMessageAmp™ II aRNA Amplification Kit, from Ambion. Aminoallyl UTPcontains a reactive primary amino group on the C5 position of uracilthat can be chemically coupled to N-hydroxysuccinimidylester-derivatized reactive dyes (NHS ester dyes), in a simple efficientreaction.

Amine-reactive quenchers are commercially available, for example QSY® 9carboxylic acid, succinimidyl ester, from Invitrogen. The real-timemicroarray technology is used with minimal modifications to the samplepreparation and labeling procedures: namely, with the simplesubstitution of the QSY-9 ester for the Cy-dye ester. In this case,total RNA samples are prepared as described above. The purified totalRNA is then amplified using the Amino Allyl MessageAmp™ II aRNAAmplification Kit, from Ambion, and the resulting aRNA is labeled withQSY9. This labeled RNA population is then used in hybridization withArabidopsis real-time microarrays. Such arrays consist of an arrangedcollection of probes that correspond to all (or a subset of) the genesin the Arabidopsis genome. The oligonucleotide probes are labeled with afluorescent moiety (such as Cy3) and printed by contact deposition ontoCodeLink slides (GE Healthcare), which are processed and blocked afterprinting following manufacturer's instructions.

Example 4

This example relates to using an algorithm to measurecross-hybridization. A variety of different techniques to recover thesignal, including, but not limited to, total least squares, ESPRIT, andProny's method, (see Dowling et. al. IEEE Trans. on Antennas andPropag., vol. 42, no. 5, 1994 and van der Veen et al. Proc. of the IEEE,81(9):1277-1308, 1993).

In this example we study the performance of one such algorithm insimulation and illustrate the results in FIG. 19. In particular, weconsider the so-called total least squares (TLS) algorithm in thesituation where two target analytes bind to the same probe spot—one dueto hybridization, and the other due to cross-hybridization. Parametersof the system (probabilities of hybridization, cross-hybridization,release, etc.) are chosen so as to mimic realistic experimentalscenarios. The probability of hybridization is assumed to be 5 timesgreater than the probability of cross-hybridization (i.e.,p_(h)/p_(c)=5). The number of hybridizing target is $n_(h)=10⁹$, whilethe number of cross-hybridizing molecules is varied. In FIG. 19, we plotthe relative mean-square error of estimating n_(h) (averaged over manyrealizations of noise) as a function of the ratio n_(h)/n_(c). Thesimulation results indicate potentially successful suppression ofcross-hybridization over 3 orders of magnitude of n_(h)/n_(c).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-66. (canceled)
 67. A system for determining at least one parameterassociated with a binding interaction between at least one analyte andat least one probe, comprising: a substrate comprising a surface havingsaid at least one probe coupled to said surface; a signal source thatprovides an input signal to said surface; a chamber in fluidcommunication with said surface, wherein during use, said chamberretains a fluid comprising said at least one analyte under conditionsthat are sufficient to permit said binding interaction between said atleast one analyte and said at least one probe; a detector that detectsoutput signals from said surface in response to said input signal fromsaid signal source, which output signals are detected at multiple timepoints in real time during said binding interaction and are indicativeof said binding interaction; and a controller in communication with saiddetector, wherein said controller (i) determines said at least oneparameter associated with said binding interaction based at least inpart on said output signals from said detector, and (ii) provides anoutput indicative of said at least one parameter.
 68. The system ofclaim 67, further comprising an electronic storage device that storessaid output.
 69. The system of claim 67, wherein said at least one probeprovides a fluorescence signal, a luminescence signal, or an absorptionsignal.
 70. The system of claim 67, wherein said at least analyteprovides a fluorescence signal, a luminescence signal, or an absorptionsignal.
 71. The system of claim 67, wherein said input signal is anelectromagnetic signal.
 72. The system of claim 67, wherein said atleast one parameter comprises: a forward binding reaction rate; abackward binding reaction rate; C which is an original quantity of saidat least one analyte in said fluid; C−(P_(o)−P(t)) which is an availableanalyte density at time t, wherein P_(o) is a number of unboundmolecules of said at least one probe, and wherein P(t) is a number ofunbound molecules of said at least one probe at time t; t which is atime constant; P_(∞) which is a steady-state value of P(t); n(t), whichis a number of analytes in said fluid that are specific to said probemolecule; P_(near), which is a probability that said at least oneanalyte is in close proximity to said probe molecule; P_(h), which is aprobability that, in a unit interval of time, said at least one analytebinds to said probe once said analyte is near; P_(r), which is aprobability that, in a unit interval of time, said at least one analytebound to said probe molecule is released; N, which is a total number ofanalyte molecules; q(t), which is a number of unbound molecules of saidat least one analyte as a function of time; a reaction rate between saidat least one analyte and said at least one probe; and across-hybridization parameter.
 73. The system of claim 67, wherein saidsubstrate comprises a plurality of different probes coupled to saidsurface.
 74. The system of claim 67, wherein said fluid is configured tocontain a plurality of different analytes, including said at least oneanalyte.
 75. The system of claim 67, wherein said at least one probe islabeled with a donor.
 76. The system of claim 67, wherein said at leastone analyte is labeled with an acceptor.
 77. The system of claim 76,wherein said acceptor is a quencher.
 78. The system of claim 67, whereinsaid at least one analyte is not labeled with a metal cluster.
 79. Thesystem of claim 67, wherein said binding interaction is non-competitive.80. The system of claim 67, wherein during use, said output signals aredetected while said fluid is in contact with said surface.
 81. Thesystem of claim 80, wherein during use, said output signals are detectedby said detector without washing said fluid from surface.
 82. The systemof claim 67, wherein said surface comprises a plurality of probe spots,and wherein a given probe spot of said plurality of probe spots includessaid at least one probe.
 83. The system of claim 67, wherein during use,said output signals are generated in the absence of fluorescentresonance energy transfer.
 84. The system of claim 67, wherein saiddetector is integrated with said substrate.
 85. The system of claim 67,wherein said signal source and said detector are disposed adjacent to asame side of said substrate.
 86. The system of claim 67, wherein duringuse, said fluid comprises a plurality of different analytes.
 87. Thesystem of claim 67, further comprising an assembly that is capable ofchanging a temperature of said fluid and/or said substrate.